KR102399971B1 - Composition for Silkworm-collagen and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a silkworm collagen health food composition and a method for producing the same, and more particularly, to silkworm collagen health containing an active ingredient of silkworm and capable of enhancing skin improvement and health promotion effect by marine collagen extracted from fish It relates to a food composition and a method for preparing the same.
The silkworm collagen health food composition of the present invention contains an enzyme-treated silkworm powder obtained by treating silkworm powder with an enzyme, and marine collagen extracted from fish.

Description

Silkworm-collagen health food composition and its manufacturing method {Composition for Silkworm-collagen and manufacturing method thereof}

The present invention relates to a silkworm collagen health food composition and a manufacturing method thereof, and more particularly, to silkworm collagen health containing active ingredients of silkworm and capable of enhancing skin improvement and health promotion effects by marine collagen extracted from fish. It relates to a food composition and a method for preparing the same.

Collagen is the most abundant protein in the animal body and accounts for about 30% or more of body protein, and it has been found that there are at least 19 types (Type I-XIX). In addition, collagen is a major protein of connective tissue of animals, supports tissues or organs, and surrounds the body surface, thereby maintaining the body shape. In particular, as a major component of connective tissue such as skin, cartilage, and bone in the living body, 40% is the skin, 20% is the bone and cartilage, and is widely distributed throughout the body such as blood vessels and internal organs.

Collagen has a triple helix structure, unlike myofibrillar proteins, which have a double helix structure, with a diameter of about 14 to 15 nm, a length of 280 to 300 nm, and an average molecular weight of about 300 KDa. It has a regular amino acid arrangement and forms a physically and biologically stable structure by covalent crosslinking within the tropocollagen molecule, the basic unit molecule of fibrous protein, or between tropocollagen molecules. .

Collagen is divided into animal collagen and marine collagen (marine collagen, fish collagen) according to the extracted raw material. Animal collagen extracted from cows, chickens, and pigs is generally widely used because it is strong against heat and easy to process. In the case of marine collagen, which is extracted from fish, it has high stability compared to animal collagen, but its absorption in the body is rather poor.

On the other hand, silkworm ( Bombyx mori L. ) is a larva of the Bombyx moth belonging to the family Bombyx mori, which can be mass-bred and has a high protein content, making it a high-protein food material (Cho, CH et al., Effect of temperature, time and pH on the extraction of proteinin a). chrysalis of silkworm, Korean J. Biotechnol. Bioeng., 4, P65-68, 1989), high in essential amino acids and n-3 polyunsaturated fatty acids (Seong, SI et al., Effect of metamorphois on the major hemolymph proteins of the silkworm, Arch. Insect Biochem. Physiol., 2, P91-104, 1985).

In addition, the sericin protein derived from silkworm mainly consists of serine, asparticacid and glycine, and it has been reported to have high hepatoprotective and antioxidant effects (Kato, N. et al., Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity, Biosci. Biotechnol. Biochem., 62, P145-147, 1998).

In addition, it has been reported that physiological activities such as antioxidant action, thrombolytic action, and inhibition of tyrosinase activity in fermented silkworms obtained by fermenting silkworm powder with high protein content with microorganisms of the genus Bacillus are increased compared to silkworm powder before fermentation. (Cha, J. Y. et al., Biological activity of fermented silkworm powder, J. Life Sci., 19, P1468-1477, 2009)

However, in the case of silkworm powder prepared only by hot air drying or freeze-drying in the prior art, there is a disadvantage in that it is difficult to obtain the effect by the active ingredient of silkworm.

Korean Patent No. 10-1636680 discloses a method for producing a low molecular weight collagen peptide derived from fish, and Korean Patent Publication No. 10-1918759 discloses a processed food using lyophilized silkworm powder and a method for manufacturing the same, As in the present invention, nothing has been revealed about the 'silkworm collagen health food composition'.

Republic of Korea Patent Publication No. 10-1636680 Republic of Korea Patent Publication No. 10-1918759

The present invention is to solve the above problems, and contains enzyme-treated silkworm powder and marine collagen to enhance health promotion effect, and the peptide derived from the enzyme decomposition product of silkworm acts to increase the absorption rate of collagen in the body. It is an object to provide a collagen health food composition.

The silkworm collagen health food composition of the present invention for achieving the above object contains enzyme-treated silkworm powder obtained by enzymatically treating silkworm powder, and marine collagen extracted from fish.

In addition, it further contains vitamin C and additives, and contains 20 to 50% by weight of the enzyme-treated silkworm powder, 5 to 40% by weight of the marine collagen, 1 to 10% by weight of the vitamin C, and 30 to 60% by weight of the additives. .

The additive is a firefly extract.

And the manufacturing method of the silkworm collagen health food composition of the present invention for achieving the above object comprises: a first step of enzymatically treating the silkworm powder to obtain an enzyme-treated silkworm powder; a second step of preparing marine collagen extracted from fish; A third step of mixing the enzyme-treated silkworm powder with the marine collagen, wherein the first step includes: a) freeze-drying the silkworm to obtain the silkworm powder having a particle size of 100 to 300 mesh; b) the preparing a silkworm suspension by suspending silkworm powder in water, c) adding a proteolytic enzyme to the silkworm suspension to hydrolyze it at 40 to 70° C. for 1 to 2 days to obtain a hydrolyzate, d) the hydrolyzate and spray-drying to obtain an enzyme-treated silkworm powder.

As the proteolytic enzyme, 0.5 parts by weight of bromelain and 1.0 parts by weight of papain are added with respect to 100 parts by weight of the silkworm suspension.

In the mixing step, vitamin C and additives are further mixed, and the additive is a firefly extract.

As described above, the silkworm collagen health food composition of the present invention contains enzyme-treated silkworm powder obtained by enzymatic treatment of silkworm powder, and marine collagen extracted from fish, and is useful for improving health, such as diabetes.

In addition, when the silkworm collagen health food composition of the present invention is ingested, the peptide of the enzyme-treated silkworm powder acts to increase the absorption rate of collagen in the body.

1 is an image showing SDS-PAGE of enzyme-treated silkworm powder according to the type of proteolytic enzyme;
Figure 2 is a table showing the cell viability evaluation experimental results,
3 is a table showing the results of the evaluation of the inhibitory activity of α-glucosidase,
4 is a table showing the results of the insulin secretion ability evaluation experiment,
Figure 5 is a graph showing the experimental results of Figure 4,
6 is a photograph showing the silkworm collagen health food composition of the present invention,
7 is a photograph showing the water solubility test result of the silkworm collagen health food composition of the present invention.

Hereinafter, a silkworm collagen health food composition and a manufacturing method thereof according to a preferred embodiment of the present invention will be described in detail.

The silkworm collagen health food composition according to an embodiment of the present invention contains enzyme-treated silkworm powder obtained by enzymatically treating silkworm powder, and marine collagen extracted from fish. For example, the silkworm collagen health food composition may be composed of 1 to 90% by weight of enzyme-treated silkworm powder, and 1 to 90% by weight of marine collagen.

Enzyme-treated silkworm powder is an enzyme-treated silkworm powder.

Silkworm ( Bombyx ) mori ) is a larva of the silkworm moth belonging to the family Silkworm moth, and it grows by eating mulberry leaves to make a strong cocoon and pupate. The body is cylindrical and consists of three parts: head, chest, and abdomen. It hates direct sunlight, and its body is milky and covered with a soft chitinous shell, giving it a soft touch. When hatched from an egg, the size of the silkworm is about 3mm, and it is hairy and has a black color. Silkworms grow while eating mulberry leaves, sleep at 4 instars, and grow rapidly when they reach 5 instars, reaching 8cm. The larval period until the end of the fifth instar is usually around 20 days. At the end of the fifth instar, they stop eating mulberry leaves and start making cocoons.

Silkworm contains 1-deoxynojirimycine (DNJ), and 1-deoxynojirimycin is effective in lowering blood sugar, antiviral and cancer cell metastasis. In addition, silkworm has various effects such as liver toxicity recovery effect and constipation improvement effect, brain function improvement effect and aging delay effect.

Enzyme-treated silkworm powder obtained by enzymatically treating silkworm powder may be formed in a particle size of 200 to 400 mesh.

Marine collagen is collagen extracted from fish. Marine collagen can be used by purchasing a commercial powder product or directly extracted from fish. For example, marine collagen may be extracted from the fish skin, which is a by-product of fish.

Marine collagen is used in powder form with a particle size of 200 to 400 mesh.

Since the silkworm collagen health food composition of the present invention contains enzyme-treated silkworm powder obtained by enzymatically treating silkworm powder, and marine collagen extracted from fish, the efficacy of silkworm and collagen can be obtained at the same time. In addition, when the silkworm collagen health food composition of the present invention is ingested, the peptide of the enzyme-treated silkworm powder acts to increase the absorption rate of collagen in the body.

In the present invention, 'food composition' means a natural product or processed product containing one or more nutrients, and preferably means a state that can be eaten directly through a certain processing process, and has a conventional meaning As such, it refers to all foods, food additives, functional foods and beverages.

Preferably, the present invention may be provided as a health food composition containing enzyme-treated silkworm powder and marine collagen. Here, 'health food composition' refers to a food group or food composition that has added added value so that it acts and expresses the function of the food for a specific purpose using physical, biochemical, and bioengineering methods, etc. It refers to food that has been designed and processed to sufficiently express the body control functions related to recovery, etc.

The health food composition according to the present invention includes, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional foods (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly products, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings Food (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), candy, chocolate, gum, ice cream, dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (eg fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), natural seasonings (eg ramen soup, etc.) are included, but not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

Furthermore, the present invention provides various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, It may contain a protective colloidal thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, etc., and the components may be used independently or in combination

In addition, the health food composition may include food pharmaceutically acceptable food supplement additives, and may further include suitable carriers, excipients and diluents commonly used in the manufacture of functional foods.

Since the health food composition can be formulated in various forms, the form is not particularly limited. Preferably, the health food composition may be formed in any one formulation selected from granules, tablets, powders, pills, and capsules. The health food composition having such granules, tablets, powders, pills, and capsules is easy to carry and easily ingested anytime, anywhere.

Preferably, the silkworm collagen health food composition of the present invention is provided in powder form and can be consumed as it is, and can be consumed as tea by adding it to cold water, hot water, or milk.

On the other hand, the silkworm collagen health food composition according to another example of the present invention may contain enzyme-treated silkworm powder, marine collagen, vitamin C, and additives. For example, the silkworm collagen health food composition may contain 20 to 50% by weight of enzyme-treated silkworm powder, 5 to 40% by weight of marine collagen, 1 to 10% by weight of vitamin C, and 30 to 60% by weight of additives.

Vitamin C is a substance that is a biosynthesis precursor of collagen, and is used as a substance for the body's own collagen biosynthesis when ingested.

The additive is to add the functionality of the silkworm collagen health food composition, and a firefly extract can be used.

Firefly ( Sanicula chinensis ) is a perennial plant belonging to the umbel family, and grows in the forest. Cham firefly is used as an herb when tender, and the rhizome is also used as a diuretic and antipyretic.

Charm body extract may be extracted from the leaves of Charm firefly. As an example of extraction, 2 to 20 times the weight ratio of the extraction solvent is added to the leaves of firefly and mixed, and then extracted by hot water extraction, cold chim extraction or hot chim extraction at 10 to 150° C. for 1 to 48 hours.

As the extraction solvent, at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof may be used. Methanol, ethanol, etc. can be used as a C1-C4 lower alcohol, and butylene glycol, propylene glycol, pentylene glycol, etc. can be used as a polyhydric alcohol. And as the mixture, a mixture of water and a lower alcohol, a mixture of water and a polyhydric alcohol, a mixture of a lower alcohol and a polyhydric alcohol, or a mixture of water and a lower alcohol and a polyhydric alcohol may be used.

After extraction, filtration and concentration under reduced pressure, a powder form of firefly extract can be obtained through freeze-drying, spray-drying, etc. Freeze-drying For example, rapidly frozen at a temperature of -50 to -40 ° C for 10 to 20 hours, then dried at about -40 ° C for 48 hours in a freeze dryer with a vacuum degree of 0.1 to 0.5 torr, and then 100 to 300 mesh particle size crushed with

And as an additive, in addition to the firefly extract, it may further include any one or more selected from the group consisting of anhydrous crystalline glucose, crystalline cellulose, L-arginine, and vitamins/mineral premix, and may further include flavoring agents, flavoring agents, and coloring agents. can

Hereinafter, a method for producing a silkworm collagen health food composition according to a preferred embodiment of the present invention will be described in each step.

1. The first step of obtaining the enzyme-treated silkworm powder and

First, the silkworm powder is treated with an enzyme to obtain an enzyme-treated silkworm powder.

The process of obtaining the enzyme-treated silkworm powder is a) freeze-drying the silkworm to obtain the silkworm powder having a particle size of 100 to 300 mesh, b) suspending the silkworm powder in water to prepare a silkworm suspension, c) It may consist of a step of adding a proteolytic enzyme to the silkworm suspension and hydrolyzing it at 40 to 70° C. for 1 to 2 days to obtain a hydrolyzate, d) spray-drying the hydrolyzate to obtain an enzyme-treated silkworm powder. .

Any edible species of silkworm can be used. It is preferable to use silkworms grown by eating Cuji mulberry leaves as silkworms. In order to obtain silkworm powder, any drying method may be applied, but it is preferable to apply a freeze-drying method to minimize changes in the composition of silkworms.

For example, live silkworms of 3 days of age 5 are rapidly frozen at -40 to 50 ° C. for 8 to 16 hours, and then dried by freezing again in a vacuum chamber at -70 ° C. for 2 to 10 hours. Thereafter, it is pulverized with a grinder to obtain silkworm powder having a particle size of 100 to 300.

Next, the silkworm powder is suspended in water to prepare a silkworm suspension. It is recommended to add 5~20ml of water per 1g of silkworm powder. When water is added to less than 5 ml, there is a problem that the enzymatic reaction is not performed properly because the silkworm suspension is not made in a liquid state.

In addition, sodium hydroxide (NaOH) solution or hydrogen chloride (HCl) solution may be added to adjust the pH of the silkworm suspension. In this case, the pH of the silkworm powder suspension can be adjusted to 5-7.

Next, a hydrolyzate is obtained by adding a proteolytic enzyme to the silkworm suspension.

Proteases Bromelain, Papain, Protamax, Trypsin, Proleather FG-f, Flavozyme, Protease A (Protease A), Aroase AP-10 (Aroase AP-10), Fiscalase (Pescalase), Protease P, Protease N, Alcalase 2.4L, Ficin (Ficin) and selected from the group consisting of Neutrase (Neutrace) Any one or more may be used. Preferably, bromelain and papain are used together as proteolytic enzymes. When bromelain and papain are used together, the decomposition effect of silkworm protein is high and the reaction is fast.

The protease may be added in an amount of 0.1 to 2 parts by weight based on 100 parts by weight of the silkworm suspension. For example, 0.5 parts by weight of bromelain and 1.0 parts by weight of papain may be added based on 100 parts by weight of the silkworm suspension.

After adding a protease to the silkworm suspension, it is stirred at 40 to 70° C. for 1 to 2 days to hydrolyze the silkworm powder. The hydrolyzate thus obtained was inactivated by heating at 90 DEG C for 30 minutes.

Next, the hydrolyzate is dried to obtain an enzyme-treated silkworm powder. As a drying method, a spray drying method may be used. Spray drying is a method of drying a liquid by spraying it in hot air. By spraying a hydrolyzate in a spray dryer, an enzyme-treated silkworm powder having a particle size of 200 to 400 mesh is obtained.

2. The second step to prepare marine collagen

Marine collagen is collagen extracted from fish. A powder product commercialized as such marine collagen is available.

In addition, marine collagen can be directly extracted from fish and used. Preferably, marine collagen is extracted from the fish skin of by-products of fish.

The fish skin is a shell of fish, and shells of halibut, eel, sea bass, red sea bream, salmon, etc. can be used.

For marine collagen extraction, a sodium hydroxide solution is added to the fish skin powder to obtain an alkali residue from which non-collagenous proteins are removed, and an acetic acid solution is added to the alkali residue and stirred, and then the supernatant is separated by a centrifuge to obtain acid-solubilized collagen. Extracting, adding pepsin to the acid-solubilized collagen and stirring, separating the supernatant with a centrifuge, and dialyzing the precipitate precipitated by adding sodium chloride solution to distilled water to extract pepsin-solubilized collagen; A purification step of refining may be performed.

First, the fish skin is washed and dried, and then pulverized to a particle size of 50 to 150 mesh to prepare a fish skin powder. You can go through a pre-treatment process to remove the scales attached to the fish skin before drying. For this purpose, the washed fish is mixed with 0.1M sodium hydroxide solution and stirred for 6 to 12 hours. When stirred with sodium hydroxide solution, there is a slight difference depending on the type of fish, but 80 to 90% of scales are easily removed.

After mixing fisheye powder and sodium hydroxide solution in a weight ratio of 1:5 to 10, stirring at room temperature (20-25°C) for 12 to 24 hours, and then using a centrifuge to obtain alkali residues from which non-collagenous proteins have been removed can do.

Then, after washing the alkali residue with distilled water, acetic acid solution is added to extract collagen. The alkali residue and the acetic acid solution are mixed in a weight ratio of 1:5 to 10, stirred at room temperature (20-25° C.) for 12 to 24 hours, and then the supernatant is separated using a centrifuge to obtain acid-solubilized collagen.

Then, pepsin is added to the acid-solubilized collagen, stirred for 10 to 20 hours, the supernatant is separated by centrifugation, and the precipitate precipitated by adding 2M sodium chloride solution is dialyzed with distilled water to obtain pepsin-solubilized collagen.

Then, to obtain marine collagen by purifying pepsin-solubilized collagen, pepsin-solubilized collagen was dialyzed against 20 mM Na2HPO4 to inactivate pepsin, and then dialyzed against a 50mM acetic acid solution (pH 4.8) containing urea, followed by fractionation using ion chromatography. elute For example, in a column filled with cellulose phosphate (P11, Whatman, Maidstone, UK), purification is performed with a linear gradient (60 ml/h) of 0 to 600 mM NaCl, and the fraction eluted at 230 nm is 0.5 containing 2.0 M NaCl. It is recovered by dialysis with M acetic acid solution, then dialyzed with distilled water and freeze-dried to obtain high-purity marine collagen. Marine collagen is used in powder form with a particle size of 200 to 400 mesh.

3. 3rd step of mixing enzyme-treated silkworm powder and marine collagen

When the enzyme-treated silkworm powder and marine collagen are prepared, the enzyme-treated silkworm powder is mixed in a ratio of 1 to 90% by weight and 1 to 90% by weight of marine collagen to finally prepare the silkworm collagen health food composition of the present invention.

In addition, the enzyme-treated silkworm powder and marine collagen may be further mixed with vitamin C and additives to prepare the silkworm collagen health food composition of the present invention. In this case, 20 to 50% by weight of the enzyme-treated silkworm powder, 5 to 40% by weight of marine collagen, 1 to 10% by weight of vitamin C, and 30 to 60% by weight of additives may be mixed.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Selection of hydrolytic enzyme>

Silkworm powder was prepared by purchasing commercially available products. 1000 ml of distilled water was added to 100 g of silkworm powder and mixed at room temperature for 30 minutes to prepare a silkworm suspension.

After preparing a total of 4 samples by adding 0.01 g each of Bromelain, Papain, Protamax, and Trypsin to 10 ml of silkworm suspension, use a shaking incubator and reacted at 50°C for 120 hours, followed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

Protein markers were purchased from Sigma (USA) and sample buffer (60mM Tris-HCl, 25% glycerol, 2% SDS, 14.4mM 2-mercaptoethanol, 0.1% bromophenol blue, 10mL distilled water) ) was dissolved in After adding the same amount of each sample and sample buffer, it was heated in water at 100° C. for 5 minutes to induce complete denaturation of the protein, followed by electrophoresis (MiniProtean, BIO-RAD Laboratories, Inc. USA.). As the buffer solution for electrophoresis, 0.025M-Trisbase-0.192 M Glycine (pH8.3) solution containing 0.1% (w/v) SDS was used, and the resulting gel was stained with Coomasie blue R-250 solution (Coomasie blue). R-250; 1.0g Coomassie blue R-250, 450mL methanol, 450mL H2O, 100mL glacial acetic acid) was used at room temperature for 2 hours with shaking. Decolorization was carried out by pouring a decolorization reagent (100mL methanol, 100mL glacialacetic acid, 800mL H2O) to lightly submerge the gel and shaking while exchanging the reagents twice for 30 minutes. The bands stained on the gel obtained at this time were compared with the protein markers and are shown in FIG. 1 .

Referring to FIG. 1 , it was confirmed that the hydrolysis effect was more excellent when using bromelain and papain compared to protamax or trypsin.

<Production of silkworm collagen health food composition>

A silkworm collagen health food composition was prepared using bromelain and papain as proteolytic enzymes.

(Example 1)

Silkworm powder and marine collagen powder were prepared by purchasing commercially available products. 1000 ml of distilled water was added to 100 g of silkworm powder, mixed at room temperature for 30 minutes, and the silkworm powder was suspended to prepare a silkworm suspension. After adding 5 g of bromelain and 10 g of papain to 1000 g of silkworm suspension, hydrolysis was performed at 60° C. for 24 hours using a shaking incubator to obtain a hydrolyzate.

Then, the hydrolyzate was put into a sterilizer and enzymatically inactivated at 90° C. for 30 minutes, and an enzyme-treated silkworm powder having a size of about 200 mesh was obtained through a spray dryer.

And enzyme-treated silkworm powder and marine collagen were mixed in a weight ratio of 7:3 to prepare a powdered silkworm collagen health food composition.

(Example 2)

The enzyme-treated silkworm powder and marine collagen used in Example 1 were mixed with vitamin C and firefly extract to prepare a silkworm collagen health food composition. That is, by mixing enzyme-treated silkworm powder 40% by weight, marine collagen 25% by weight, vitamin C 5% by weight, anhydrous crystalline glucose 20, and firefly extract 10% by weight, a silkworm collagen health food composition was prepared.

Firefly extract was obtained by adding 8 times the weight ratio of water as an extraction solvent to the leaves of Firefly, followed by extraction at 90° C. for 6 hours, filtered through filter paper, and then freeze-dried to obtain a powder having a particle size of 150 mesh.

<Sample preparation>

To the silkworm collagen health food composition of Example 1, 80% ethyl alcohol was added in a ratio of 1:4 (w/v) and shaken at 150rpm for 24 hours at room temperature. Thereafter, the precipitate was removed by centrifugation at 3,000 rpm for 20 minutes. This was again filtered by using a syringe filter to prepare a sample to be used for the following experiments.

<Cytotoxicity test>

A cytotoxicity test was performed on the RIN-m5F cells used in the insulin secretory ability evaluation experiment.

The RIN-m5F cell line was purchased from the American type culture collection (ATCC, USA) and cultured at 37° C. and 5% CO ₂ condition using RPMI-1640 medium containing 10% fetal bovine serum (FBS). Then, the RIN-m5F cell line was dispensed in a 96-well culture plate at a concentration of 2 x 10 ⁴ cells/well, and the sample was cultured for 24 hours using RPMI-1640 medium containing 10% fetal bovine serum (FBS) to evaluate the concentration. The test substance was prepared by diluting it with

The test substance was incubated for 24 hours, and MTT solution was added so that the final concentration was 0.5 mg/mL, and then reacted for 4 hours. After removing the medium, cell lysis was performed using 100 μL of DMSO, and absorbance was measured with triplicate (3 wells) at 540 nm using a microplate reader (Molecular device). 2 shows the experimental results.

2, as a result of evaluating cell viability according to concentration, 4.7±1.9%, 8.5±44%, 10.2± at 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02 and 0.01% 4.3% 0, 72.4±7.9%, 90.5±3.2%, 98.6±2.4%, 98.6±6.5%, 105.9±7.2%, 91.8±3.0%, 97.7±7.1%, and 110.0±4.9% were identified as about 0.63% concentration No cytotoxicity was observed.

<Anti-diabetes experiment>

For the antidiabetic test of the silkworm collagen composition of the present invention, an experiment was conducted to evaluate the in-vitro antidiabetic effect on the inhibitory activity of α-glucosidase and the insulin secretion ability.

1) α-glucosidase inhibitory activity evaluation experiment

The following experiment was conducted to evaluate the inhibitory activity of α-glucosidase for the antidiabetic experiment.

Samples were prepared at concentrations of 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024 and 0.0% using 50 mM potassium phosphate buffer (pH6.8). To 50 μL of each concentration test substance, 100 μL of 2.5 mM p-nitrophenyl α-D-glucopyranoside (pNPG) was added, and 50 μL of α-glucosidase (Sigma, USA) was added and reacted at 37 ° C. The degree of inhibition of α-glucosidase activity was measured with triplicate (3 wells) through the reaction rate obtained by measuring the absorbance of -nitrophenol at 405 nm for 10 minutes at 1 minute intervals. Acarbose was used as a positive control material, and acarbose (Sigma, USA) was treated in 50 mM potassium phosphate buffer (pH6.8) to evaluate each concentration. 3 shows the experimental results.

3, as a result of measuring the α-glucosidase activity inhibitory ability according to the concentration of the experimental group of the present invention, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024 and 0.0% were respectively 96.4. ±0.3%, 93.4±1.4%, 91.9±1.1%, 79.4±0.9%, 68.9±1.8%, 55.6±1.6%, 41.2±1.3%, 29.6±0.9%, 21.7±1.7%, 19.1±1.2%, 16.5 It was confirmed as ±0.9% and 0.0 ±0.0%, and the concentration that inhibited α-glucosidase by 50% was confirmed to be about 0.6%.

2) Insulin secretion ability evaluation experiment

As an insulinoma cell line derived from a mouse pancreatic cell tumor, an insulin secretory ability evaluation experiment was conducted using the RIN-m5F cell line, a test system widely used in studies to evaluate the insulin secretion ability of antidiabetic cells.

The RIN-m5F cell line was purchased from the American type culture collection (ATCC, USA) and cultured at 37° C. and 5% CO ₂ condition using RPMI-1640 medium containing 10% fetal bovine serum (FBS). The RINm5F cell line was aliquoted into a 48 well culture plate at a concentration of 5 x 10 ⁵ cells/well and cultured for 24 hours. Cells in each well were treated with 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM NaHCO3, 10 mM HEPES-free acid, 1 g/L bovine serumalbumin, and 1.1 mM D-glucose. Washing was repeated 3 times for 5 minutes each with Kreb's Ringer Buffer (pH 7.4). In Kreb's Ringer Buffer containing 5.6mM D-glucose, 500 μL of each sample was placed in each well with cells and reacted for 1 hour. The reaction solution, the supernatant, was taken and insulin was analyzed. Insulin concentration was measured in triplicate (3 wells) using the Morinaga UltraSensitive Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Japan). As a positive control, glibenclamide was treated at a single concentration of 5 μg/mL and the same evaluation was performed. 4 to 5 show the experimental results.

4 to 5 , the insulin secretion ability was evaluated at a concentration without cytotoxicity in the experimental group. As a result of the evaluation, the vehicle group was measured to be 3.08±0.10 ng/mL, and the positive control, glibenclamide 5ug/mL, was 4.36±0.17 ng/mL, a significant increase compared to the vehicle group. The experimental group was 4.22±0.10 ng/mL, 4.62±0.26 ng/mL, 5.05±0.09 ng/mL, 5.41±015 ng/mL and 5.62±0.45 ng/mL at 0.04, 0.08, 0.15, 0.3 and 0.6%, respectively. As a result, a significant increase was confirmed at all concentrations compared to the vehicle group.

When the above-mentioned antidiabetic experimental results are taken together, it was confirmed that the silkworm collagen health food composition of the present invention increased insulin secretion with the inhibitory effect of α-glucosidase at a specific concentration.

<Water solubility test>

The silkworm collagen health food composition prepared in Example 2 is provided in the form of green powder as shown in FIG. 6 . In order to check whether the powdered silkworm collagen health food composition dissolves well in water, the following water solubility test was performed.

A total of 4 test tubes were prepared in which 0.1 g, 0.5 g, 1 g, and 15 g of the silkworm collagen health food composition was added to 10 ml of water, respectively. After stirring the contents of each test tube well with a glass rod, the appearance observed with the naked eye is shown in FIG. 7 .

It can be seen that the silkworm collagen health food composition of the present invention is in powder form and is relatively soluble in water.

<Antibacterial test>

In order to examine the antibacterial effect, sterile water was added to the health food composition of Examples 1 and 2, kneaded, and then left at room temperature for 10 days, and then the number of general bacteria was measured. To measure the number of bacteria, 5 g of dough sample was collected, transferred to a sterile homogenizer, and mixed in 45 mL of 0.5% peptone solution for 100 seconds. This solution was again sequentially diluted with 0.5% peptone solution, and 0.1 mL of the diluted solution was spread on a plate count agar and incubated at 35° C. The results are shown in Table 1 below. The first test piece is Example 1, and the second test piece is Example 2.

Test group 1 (CFU/ml) Test group 2 (CFU/ml) 1.8×10 ⁵ 4.2×10 ²

Looking at the results of Table 1, it was confirmed that the second test group to which the firefly extract was added effectively inhibited the growth of microorganisms compared to the first test group.

As mentioned above, although the present invention has been described with reference to the embodiments, these are merely exemplary, and those of ordinary skill in the art will understand that various modifications and equivalent embodiments are possible therefrom. Accordingly, the true protection scope of the present invention should be defined only by the appended claims.

Claims (6)

delete delete delete A first step of enzymatically treating the silkworm powder to obtain an enzyme-treated silkworm powder;
a second step of preparing marine collagen extracted from fish;
A third step of mixing 20 to 50% by weight of the enzyme-treated silkworm powder, 5 to 40% by weight of the marine collagen, 1 to 10% by weight of vitamin C, and 30 to 60% by weight of additives;
The first step is a) freeze-drying the silkworm to obtain the silkworm powder having a particle size of 100 to 300 mesh, b) suspending the silkworm powder in water to prepare a silkworm suspension, c) sodium hydroxide solution or Adjusting the pH of the silkworm suspension to 5 to 7 using a hydrogen chloride solution, and then hydrolyzing at 40 to 70° C. for 1 to 2 days by adding a proteolytic enzyme to obtain a hydrolyzate, d) spraying the hydrolyzate and drying to obtain an enzyme-treated silkworm powder,
The additive is a firefly extract,
The method for producing a silkworm collagen health food composition, characterized in that the extract is a powder obtained by adding 8 times the weight ratio of water to the firefly leaf, followed by extraction at 90° C. for 6 hours, followed by filtration and freeze-drying. The method according to claim 4, wherein 0.5 parts by weight of bromelain and 1.0 parts by weight of papain are added as the proteolytic enzyme based on 100 parts by weight of the silkworm suspension. delete

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