KR20190060511A - A composition for antioxidating comprising extracts of fermented tenebrio molitor - Google Patents

Abstract

The present invention relates to a fermented mealworm extract and to an antioxidant composition containing the same, wherein mealworm larvae are extracted using an organic solvent so as to remove oil and fat components contained in the mealworm larvae, then freeze dried, fermented using yeast strains, and then extracted using water or an organic solvent so as to obtain an extract. The fermented mealworm extract of the present invention has the advantageous effect that the total polyphenol, flavonoid, tannin and essential amino acid content is increased and changed, and yeast protein used by mealworm larvae is generated, while antioxidant activity is increased. Consequently, a composition containing the fermented mealworm extract of the present invention can be used as an antioxidant composition.

Description

TECHNICAL FIELD [0001] The present invention relates to an antioxidative composition comprising a fermented extract of brown bean curd refuse as an active ingredient,

The present invention relates to a composition for antioxidation containing a fermented brown duck extract, and more particularly to a fermented soybean oil comprising a total amount of polyphenols, flavonoids, tannins and essential components To an antioxidative cosmetic composition, a pharmaceutical composition and a food composition containing the fermented extract as an active ingredient.

The human skin consists of the epidermis, dermis, and connective tissue including the stratum corneum, and the stratum corneum is composed of a dead cell layer formed through the differentiation process of keratinocyte, the basal cell of the epidermis And is responsible for protecting the human body from the effects of the external environment. There are two major researches on the causes of skin aging. One is "internal aging", which is caused by changes in the function of cells, which are constitutive units of skin, with age, and the other is "external aging" Which can be divided into external environment such as ultraviolet rays, pollution, stress and the like.

As the cells of the skin are exposed to oxidative stress by reactive oxygen species generated in a continuous biochemical reaction for the necessary energy supply in the human body in order to supply necessary energy in vivo, the cellular components such as lipid, protein, It changes carbohydrate and DNA oxidative damage and enzymatic activity, causing various diseases such as skin cancer, and promotes aging. In addition, the biggest cause of external aging is photoaging due to ultraviolet rays, which causes acute and chronic skin injuries such as skin cancer through continuous exposure to UV.

Active oxygen introduced from the outside of the living body or generated in the living body causes many problems such as promoting aging of the living body or generating cancer. Therefore, the development and research of antioxidants that inhibit oxidation by active oxygen have been performed. Antioxidants are widely distributed in copper and plants. Many phenolic compounds, flavonoids, tocopherols, vitamin C and selenium are known in fruits and vegetables. However, the antioxidant substances present in nature can not be expected to have practically sufficient effects in skin application. Therefore, although synthetic antioxidants having excellent antioxidant ability and low cost are widely used, their use is restricted due to safety concerns such as human side effects.

Therefore, in the fields of cosmetics and foods, research and development of functional products having antioxidative effects are actively carried out, and studies using natural materials have been continuing in order to avoid toxicity or irritation to the skin.

On the other hand, the "Tenebrio molitor" is an insect of the beetle neck, about 15 mm in length, dark brown and glossy. The larvae of brown ducks are called mealworms and feed largely as food insects or pets. The reason why the wheat worms are used for pets is that they are relatively clean because they have a relatively short period of transformation and are very clean insects that feed on grain to be. Larvae moult 9-20 times depending on food and temperature until they become pupae. It becomes a pupa and becomes alien to adult after 2 ~ 3 weeks. It becomes light brown or gradually black when it is first allegory. Adults are nocturnal, hiding in the hole during the daytime and active at night. It grows in pupa and adults in spring when it swallows larvae in the grain bag near the main island.

These brown goats are reported to be highly effective as protein donors because they have the highest protein content and insufficient fat content among insects. They are known to exhibit increased anticancer activity when treated with hepatocellular carcinoma cells, in particular with anticancer substances.

Korean Patent Registration No. 10-1752253 discloses an insecticidal gum composition using a brown gurrier and a method for producing the same. Korean Patent Registration No. 10-1746111 discloses a method for preparing a gum-based food containing brown gurd And Korean Patent No. 10-1651908 discloses a composition for preventing or treating diabetes comprising an effective amount of a brown larva or an extract thereof as a patent related to the use of brown gourd, 10-2016-0041110 discloses a composition for the prevention or treatment of rheumatoid arthritis, which comprises a suspension of brown goats as an active ingredient. Korean Patent Laid-Open Publication No. 10-2014-0134331 discloses a composition for preventing or treating rheumatoid arthritis, Of the active ingredient of the suspension of obesity prevention or Korean Patent Laid-Open Publication No. 10-2015-0050712 discloses an anticancer composition comprising an extract of brown gruel or a fraction thereof as an active ingredient, Korean Patent Registration No. 10-1424125 discloses a composition for treating cancer, A composition for the treatment of inflammatory diseases including larva was reported. Korean Patent Registration No. 10-1404566 has reported a composition for preventing or treating dementia including brown goat extract.

However, there has been no report on the antioxidative effect using brown goats.

Therefore, the inventors of the present invention have continued to develop new natural substances having antioxidative effects and fewer side effects on human body. As a result, they found that the brown duck fermented extract showed excellent antioxidative effects without cytotoxicity, Completed.

Accordingly, a technical problem to be solved by the present invention is to provide a substance having an antioxidative effect and excellent human stability.

In order to solve the above-mentioned technical problem, in the present invention, a brown larva is extracted with an organic solvent to remove the fat and fat components contained in the larva and then lyophilized, followed by fermentation with a yeast strain, , Flavonoids, tannins, and essential amino acids, which are characterized in that they are obtained by extracting the extracts of the present invention by using the extracts of the present invention.

In addition, the present invention provides an antioxidative composition comprising the above-described brown gourd fermented extract as an active ingredient.

Preferably, in the present invention, the composition comprising the brown frozen fermentation extract may be a cosmetic composition, a pharmaceutical composition or a food composition.

In the present invention, the "brown duck fermented extract" is obtained by extracting brown goat larvae with an organic solvent, removing the fat and fat components contained in brown goat larvae, freeze-drying, fermenting them with yeast strains, Can be obtained by extraction with an organic solvent.

According to a specific embodiment of the present invention, the removal of the fat and fat components contained in the brown gully is carried out in an organic solvent such as water, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, Butyl alcohol, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, 30 hours, preferably 23 to 25 hours.

According to a specific embodiment of the present invention, the fermentation is carried out by inoculating a yeast strain into a brown duck larva which has been subjected to fat and oil removal, and then inoculated at 20 to 30 DEG C, preferably at 23 to 27 DEG C for 60 to 80 hours, For 70 to 75 hours. If the fermentation temperature is lower than 20 ° C, the fermentation is not sufficiently carried out or it takes a long time to ferment, which is inefficient. If the fermentation temperature exceeds 30 ° C, the activity of the yeast strain may be lowered and the fermentation may not sufficiently take place. Also, when the fermentation time is less than 60 hours, the fermented brown duck fermented extract can not sufficiently exhibit the antioxidative effect because the fermentation of the brown duck does not sufficiently take place. If the fermentation time exceeds 80 hours, And there is no difference in fermentation efficiency.

The yeast strains include, for example, strains of Saccharomyces cerevisiae , S. ellipsoideus , S. eubayanus , Saccharomyces spp . S. pastorianus , S. florentinus and the like, and Saccharomyces cerevisiae strain is preferably used.

According to a specific embodiment of the present invention, the extraction of the brown frozen fermented product is carried out in water or in an organic solvent such as water, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, Extraction with hot water, cold extraction, ultrasonic extraction, reflux cooling extraction using dichloromethane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof . Specifically, in the present invention, the brown gruel fermented product can be heated, unheated or cold-extracted using water or 60 to 100% alcohol.

In addition, the brown gully fermentation extract may be obtained by a conventional purification process in addition to the extraction method using the above extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, A brown goat fermented extract can also be obtained through fractionation.

The brown gruel fermentation extract of the present invention may be any extract, fraction, purified product, diluted solution, concentrate or dried product obtained in each step of extraction, fractionation or purification (separation, fractionation).

In the present invention, the above-mentioned " active ingredient " means a component that exhibits the desired activity alone or can exhibit activity together with a carrier which is itself inactive.

The composition of the present invention may contain from 0.005 to 50% by weight, more preferably from 0.01 to 30% by weight, and most preferably from 0.1 to 10% by weight, based on the total weight of the composition, of a brown dumpling fermentation extract. If the content of the fermented extract is less than 0.005% by weight, the antioxidant effect of the present invention can not be obtained. If the content of the fermented extract is more than 50% by weight, the effect may not be proportional to the content, There is a problem that the stability of the shape is not ensured.

The composition containing the fermented extract of the present invention has an antioxidative effect and is almost free from cytotoxicity as a natural substance.

According to one embodiment of the present invention, there is provided a cosmetic composition comprising as an active ingredient an extract of a fermented brown gruel.

The cosmetic composition according to the present invention may contain, in addition to the brown goat's fermented extract as an active ingredient, conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, Can be added.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, use is made of an animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the present invention is a powder or a spray, tosse, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Castellulose, aluminum metahydroxide, bentonite, agar or tracert, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

According to one embodiment of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient an extract of fermented brown gruel.

The pharmaceutical composition according to the present invention includes a pharmaceutically acceptable carrier in addition to the brown frozen fermentation extract. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by parenteral administration, more preferably topical application by application.

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. When the composition is an external preparation, it is preferably applied in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day, and continued for 1 month or more. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art, Into the container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to one embodiment of the present invention, there is also provided a food composition comprising an extract of fermented brown gruel as an active ingredient.

The food composition according to the present invention may further contain, as an active ingredient, a brown dough fermented extract as well as a component that is ordinarily added in the manufacture of food such as proteins, carbohydrates, fats, nutrients, seasonings and flavors .

Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

For example, when the food composition of the present invention is prepared as a drink, it additionally contains citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, etc., .

On the other hand, the brown gourd fermented extract of the present invention is a natural substance which is harmless to the human body and has little toxicity and side effects, so that it can be safely used even in long-term use and can be safely applied to cosmetic, pharmaceutical and food compositions as described above have.

Thus, the brown duck fermented extract of the present invention has an effect of increasing and changing the total polyphenols, flavonoids, tannins, and essential amino acid contents, producing brown duck larvae yeast protein, and increasing antioxidant activity. Therefore, the composition can be used as an antioxidant composition containing the fermented extract of brown gourd according to the present invention.

Figure 1 is a schematic representation of a process for the production of a brown gruel fermented extract.
FIG. 2 is a graph showing the results of measurement of the electron donating ability of the brown goat fermentation extract. FIG.
FIG. 3 shows the results of ABTS cation radical scavenging ability of brown goat fermented extract Fig.
Figure 4 shows the Fe ³⁺ reductivity Fig.
FIG. 5 is a graph showing the results of FRAP analysis of a brown duck fermented extract of a brown duck fermented extract.
FIG. 6 is a graph showing the results of Cu ²⁺ reduction (CUPRAC) measurement of a brown goat fermentation extract of a brown goat fermentation extract.
FIG. 7 is a graph showing the results of measurement of Fe ²⁺ chelation of a brown goat fermented extract of a brown goat fermentation extract. FIG.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Preparation of Brown Goose Fermented Extract

(1) Elimination of fatty substances contained in brown duck

The brown larvae were repeatedly extracted three times for 24 hours at 24 ° C with hexane and filtered with a Wattman No.2 paper filter to separate the oil and fat components. Brown goat larvae, which had been preserved and removed the fatty components, were concentrated using a rotary vacuum concentrator and lyophilized.

(2) Fermentation

The brown and larva larvae which had been removed from the oil and fat were pulverized.

Saccharomyces S. cerevisiae strain (KCTC 17299) was purchased from the Korean Cell Line Bank for the third time.

The peptone in the composition of yeast peptone dextrose (YPD) medium (1000 mL) was replaced with peptone, and the powdered brown dung larvae powder was used, and the subcultured Saccharomyces cerevisiae cerevisiae strain (KCTC 17299) was inoculated in an amount of 10 쨉 l and fermented at 25 째 C for 72 hours at a stirring speed of 100 rpm.

(3) Extraction

The fermented brown dung larva fermentation broth was subjected to reflux extraction by four methods such as hot water, cold water, 70% alcohol and 70% ethanol.

In test group 1, 1 L of brown goat larva fermentation broth was added with 1 L of water, followed by hot water extraction at 80 ° C. Separation of supernatant and precipitate was repeated twice for 2 hours. In test group 2, 1 L of brown goat larva fermentation broth was added with 1 L of water The test group 3 was extracted with 1 L of brown goat larva fermentation broth instead of water at room temperature, and in test group 4, 1 L of brown goat larva fermentation broth was replaced with 70% Test group 5 was extracted by the same method as hot water extraction with 1 L of brown goat larva fermentation broth using water instead of water.

The obtained extracts, the fermentation broth, and the culture broth were filtered using a Wattman No. 2 paper filter, and then concentrated using a rotary vacuum concentrator. The concentrated samples were filtered using a sterile membrane filter (0.45 μm) and stored at -80 ° C.

Figure 1 is a schematic representation of a process for the production of a brown gruel fermented extract.

All the experiments were repeated with 3 repetitions and the measured values were expressed as mean ± standard deviation. The statistical significance of the test results was determined by Student's t-test and statistically significant when the p value was less than 0.05.

≪ Test Example 1 > Measurement of the active ingredient content of the fermented brown goat extract

(1) Condensation type tannin content measurement

The total tannin content was determined by adding 2 mL of vanillin dissolved in H ₂ SO ₄ solution to 1 mL of the diluted sample. The absorbance was measured at 500 nm after being left for 15 minutes. The total standard tannin content of the extract was expressed as μg TE (Tannin equivalent) / g after the standard calibration curve was prepared using tannin as a reference material.

(2) Total polyphenol content measurement

The total amount of polyphenol was determined by adding 75 μl of distilled water and 25 μl of Folin-Ciocalteu phenol reagent reagent to 25 μl of the sample diluted in distilled water, reacting for 6 minutes, adding 100 μl of saturated solution Na ₂ CO ₃ , After the absorbance was measured at 765 nm, the content of polyphenol was calculated by comparing the absorbance of the standard curve with a standard curve of gallic acid.

(3) Measurement of total flavonoid content

The total flavonoid content was 1.25 mL and 75 ㎕ of 5% NaNO ₂ solution was added and left for 5 minutes. 150 μl of a 10% AlCl ₃ .6H ₂ O solution was added and the mixture was allowed to stand for 6 minutes. 500 μl of 1 M NaOH and 275 μl of distilled water were added to the reaction solution, and the absorbance at 510 nm was measured. After the standard calibration curve was prepared using catechin as the reference material, the total flavonoid content of the extract was expressed in μg CE (catechin equivalent) / g.

The results are shown in Table 1 below. As a comparative group 1, a fermentation broth fermented by inoculating Saccharomyces cerevisiae strain (KCTC 17299) subcultured in a yeast peptone dextrose (YPD) medium was used, and as a comparative group 2 Brown goat larva fermentation broth was used without further extraction.

Name of sample Polyphenol (GAE) Flavonoid (QE) Tannin (CE) Comparison group 1 27.55 + - 0.56 16.05 ± 0.22 6.50 ± 0.13 Comparison group 2 29.45 + 0.81 20.15 + 1.38 7.44 0.11 Test group 1 15.23 + - 0.14 15.35 + 1.35 6.68 + 0.03 Test group 2 24.86 ± 0.27 15.15 ± 0.76 6.61 + 0.11 Test group 3 27.86 ± 0.29 15.73 + - 0.38 7.02 ± 0.05 Test group 4 26.60 ± 0.45 15.67 ± 1.35 7.65 + 0.03 Test group 5 29.40 ± 0.01 17.72 ± 0.22 7.47 ± 0.03

In Table 1 above, GAE is the milligrams gallic equivalent per gram, QE is the milligram quercetin equivalent per gram, and CE is the milligram tannin equivalent per gram.

As shown in Table 1, the total polyphenol, flavonoid, and condensed tannin content were measured. As a result, it was confirmed that the most recovery was achieved by using the alcohol in the method of extracting the fermented product.

≪ Test Example 2 > Measurement of electron donating ability of fermented brown goat extract

The electron donating ability (EDA) was measured by the Blois method. 60 쨉 l of 0.45 mM 1,1-diphenyl-2-picrylhydrazide (DPPH) was added to 120 쨉 l of each sample solution, left for 15 minutes, and absorbance was measured at 517 nm. The electron donating ability was expressed by the absorbance reduction rate of the sample solution addition group and the no addition group.

FIG. 2 is a graph showing the results of measurement of the electron donating ability of the brown goat fermentation extract. FIG. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, , And 8 represents vitamin C (ascorbic acid) as a positive control.

As shown in FIG. 2, the electron donating ability tended to increase with increasing concentration of test group, and the highest activity was observed in the brown goat fermented hot water extract (Test group 1) and 70% alcohol extract (test group 5) And also showed very good electron donating ability when compared to the positive control group. In particular, at the concentration of 1000 μg / mL, the same activity as the 10 μg / mL concentration of the positive control vitamin C was shown by the brown goat fermented hot water extract (test group 1) and 70% alcohol extract (test group 5). In addition, other brown goat fermented extracts showed higher activity than the fermentation broth.

≪ Test Example 3 > Measurement of ABTS cation radical scavenging ability of the brown goat fermented extract

Antioxidant activity using ABTS radical was measured by ABTS + cation decolorization assay. After mixing ABTS + with 7 mM 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) and 2.45 mM potassium persulfate, the mixture was allowed to stand at room temperature for 24 hours to form ABTS + 100 μl of the sample was added and left for 7 minutes, and the absorbance was measured at 734 nm.

FIG. 3 shows the results of ABTS cation radical scavenging ability of brown goat fermented extract Fig. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, , And 8 represents vitamin C (ascorbic acid) as a positive control.

As shown in FIG. 3, the cation radical scavenging ability tended to increase with increasing concentration in all samples, and showed excellent electron donating ability even when compared with the positive control group. In addition, the efficacy of the fermentation broth and fermented extract was higher than that of the strain.

≪ Test Example 4 > ³⁺ Reduction power measurement

Fe3 ⁺ → Fe2 ⁺ reduction power was measured by modifying Oyaizu's method. 1 mL of each sample was added with 1 mL each of 200 mM phosphate buffer solution (pH 6.6) and 1% potassium ferricyanide, and the mixture was reacted for 20 minutes in a water bath at 50 ° C. Then, 1 mL of 10% TCA solution Followed by centrifugation at 13,500 xg for 15 minutes. The supernatant was then mixed with 1 mL of distilled water and ferric chloride, respectively, and absorbance was measured at 700 nm.

Figure 4 shows the Fe ³⁺ reductivity Fig. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, , And 8 represents vitamin C (ascorbic acid) as a positive control.

As shown in FIG. 4, the Fe ³⁺ reducing power tended to increase as the concentration increased. Among the brown dough fermented extracts, the ethanol extracts showed high activity, and 70% Showed a slightly higher reducing power. At the concentration of 1000 ㎍ / mL, hot water and alcohol extracts showed about twice the reducing power than the fermented water.

≪ Test Example 5 > FRAP analysis of the brown goose fermented extract

(V / v) solution of 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) dissolved in 40 mM HCl and 20 mM FeCl ₃ .6H ₂ O in 300 mM acetate buffer (pH 3.6) v / v) and then warmed in a 37 ° C water bath. 100 μl of the extracts were mixed in this order, reacted at 37 ° C for 4 minutes, and the absorbance was measured at 593 nm using a UV spectrometer.

FIG. 5 is a graph showing the results of FRAP analysis of a brown duck fermented extract of a brown duck fermented extract. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, , And 8 represents vitamin C (ascorbic acid) as a positive control.

As shown in FIG. 5, by measuring the reducing power of Fe ³⁺ by other methods, it showed an activity pattern similar to the reducing power of Fe ³⁺ → Fe ²⁺ . The reducing activity increased with increasing concentration. The ethanol extracts showed higher activity than the brown dough fermented extracts at the concentration of 1000 ㎍ / mL. Unlike the previous method, the hydrothermal extract showed similar activity to the fermented product.

≪ Test Example 6 > Cu ²⁺  Reducing power (CUPRAC) measurement

The measurement of the reducing power was performed by Apak et al. (2006) was modified and measured. CuCl ₂ solution to the sample 0.25 mL (0.25 mL, 0.01 M ), a solution of ethanol four oak Pro (ethanolic neocuproine solution) (0.25 mL , 7.5 X 10 3 M) and NH ₄ Ac buffer (0.25 mL, 1.0 M), H ₂ O were added in this order, and the reaction was carried out for 30 minutes, and the absorbance at 450 nm was measured.

FIG. 6 is a graph showing the results of Cu ²⁺ reduction (CUPRAC) measurement of a brown goat fermentation extract of a brown goat fermentation extract. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, , And 8 represents vitamin C (ascorbic acid) as a positive control.

As shown in FIG. 6, the reducing power was increased as the concentration of Cu ²⁺ was increased. In the case of the brown dough fermented extract, the fermentation product and the fermented product at the concentration of 1000 μg / 70% of the extracts from the fermented extract showed a slightly higher reducing power. Also, it showed higher reducing power than 100 ㎍ / mL when compared with the positive control group, vitamin C.

≪ Test Example 7 > ²⁺  Chelating evaluation

Measurement of Fe ²⁺ chelation was performed as described in Dinis et al. (1994) was modified and measured. To 0.4 mL of the sample was added FeCl ₂ solution (0.1 mL, 0.6 mM) and bipyridyl solution (0.1 mL, 5 mM) in that order, reacted for 10 min, and absorbance was measured at 562 nm.

FIG. 7 is a graph showing the results of measurement of Fe ²⁺ chelation of a brown goat fermented extract of a brown goat fermentation extract. FIG. Where 1 is the comparison group 1, 2 is the comparison group 2, 3 is the test group 1, 4 is the test group 2, 5 is the test group 3, 6 is the test group 4, And 8 represents EDTA as a positive control.

As shown in Fig. 7, the effect of Fe ²⁺ chelation was insignificant. This is mainly due to the tendency to reverse the reducing power, so that it is expected to exhibit a specific effect on reducing power and radical scavenging ability.

Test Example 8 Amino acid analysis

(1) Analysis of constituent amino acids

Amino acid analysis was analyzed by HPLC (Agilent Technologies 1200 Series FLD) after digestion and derivatization according to the method of Daniel and Steven. Put 0.5 g of sample in a test tube, add 10 mL of 6 N HCl, and dissolve the test tube to make an ampoule. After sealing, autoclave is hydrolyzed at 110 ° C for 24 hours. Break the ampoule and filter it with filter paper. Concentrate under reduced pressure and use 5 mL of 20 mM HCl. After filtration with a 0.45-μm membrane filter, a certain amount of the filtrate was collected and analyzed by HPLC using AccQ-Tag reagent. The content was calculated by an external standard method using an integrator. The analysis conditions are shown in Table 2 below, and the results are shown in Table 3.

(2) Free amino acid analysis

Free amino acid analysis was performed by Ohara and Ariyosh 's method. In other words, 25 mg of sulfa salicylic acid was added to 10 mL of the filtrate obtained by treating 5 g of the sample in the same manner as the pretreatment per glass, and the mixture was allowed to stand at 4 ° C. for 4 hours and centrifuged (50,000 rpm, 30 min) The supernatant was filtered through a 0.45 μm membrane filter, and a certain amount of the filtrate obtained was subjected to derivatization using AccQ-Tag reagent and analyzed by HPLC. The content was calculated by an external standard method based on an integrator. The analysis conditions were the same as the constituent amino acids. The results are shown in Table 4 below.

Analysis of the constituent amino acids was carried out using the three samples (Comparative group 1, Test group 1 and Test group 5). The results were highest in test group 1, and the highest in free amino acid group. In addition, the target amino acid was increased overall, with histidine, valine, isoleucine, leucine and lysine increased. These amino acids (leucine, isoleucine, valine, etc.), which show beneficial activity in the body, help to improve muscle strength by preventing muscle damage caused by excessive exercise and muscle cells by enhancing muscle strength, and isoleucine, glutamine, So you can get a bigger workout effect after taking the amino acid before you work out. In addition, various activities such as immunity enhancement were reported, and the free amino acid content of the brown duck fermented extract of test group 5 was confirmed to be a functional material.

As a result, the antioxidative activity and amino acid changes of the products obtained through the development of the brown dough fermented products and extracts using yeast were examined. As a result, it was confirmed that the activity was increased by adding brown gruel during fermentation, The extracts of the group 5 showed the highest activity after fermentation.

It can be expected that there is a correlation between the fermentation of the brown gyoza in the test group 5, which has high free amino acid content, and the antioxidative activity of the fermented ginseng extract, indicating that it can be used as a good material such as fatigue recovery do.

Claims (4)

Characterized in that the brown goat larva is extracted with an organic solvent to remove the fat and fat components contained in the brown goat larva, followed by lyophilization, fermentation using a yeast strain, and extraction with water or an organic solvent. Phenol, flavonoid, tannin and an essential amino acid-rich brown duck fermented extract. The method according to claim 1,
Wherein the yeast strain is a strain of Saccharomyces cerevisiae . A composition for antioxidation comprising the brown gourd fermented extract according to claim 1 or 2 as an active ingredient. The method of claim 3,
Wherein the brown frozen fermentation extract is contained in an amount of 0.005 to 50% by weight based on the total weight of the composition.

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