WO2013058594A2 - Method for preparing a fermented deer antler extract having an anti-inflammatory activity, extract obtained by the method and use of such extract - Google Patents

Method for preparing a fermented deer antler extract having an anti-inflammatory activity, extract obtained by the method and use of such extract Download PDF

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WO2013058594A2
WO2013058594A2 PCT/KR2012/008588 KR2012008588W WO2013058594A2 WO 2013058594 A2 WO2013058594 A2 WO 2013058594A2 KR 2012008588 W KR2012008588 W KR 2012008588W WO 2013058594 A2 WO2013058594 A2 WO 2013058594A2
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antler
extract
complex
fermentation extract
aspergillus
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PCT/KR2012/008588
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French (fr)
Korean (ko)
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WO2013058594A3 (en
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정창휘
최학주
김동희
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(주)메레데코리아
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Priority to CN201280058853.XA priority Critical patent/CN104105492B/en
Publication of WO2013058594A2 publication Critical patent/WO2013058594A2/en
Publication of WO2013058594A3 publication Critical patent/WO2013058594A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment

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  • the present invention relates to a method for preparing an antler extract, an extract obtained therefrom and the use of such an extract, and more particularly to a method for producing an antler extract using fermented extract, an extract obtained therefrom and the use of such an extract.
  • Deer antler usually refers to the horns of a deer. Specifically, it refers to a male's horn that is slightly soft and covered with hairs. In the fall, the horns that have been softened are keratinized, which is for the fight to occupy females during the estrus, and these keratinized horns are less effective than antlers. This greenery misses the harvest time, becomes calcium, hardens, and then falls on its own.
  • antler As a method of taking antler used in traditional Chinese medicine, antler is mixed with various kinds of herbal medicines, pulverized, heated and extracted with water as a solvent to take the filtrate, and the herbal medicines are pulverized and powdered. ( ⁇ ) How to take is mainly used.
  • the above two methods have advantages and disadvantages.
  • the extraction and taking are easy to absorb the active ingredient of the antler, while the active ingredient contained in the extract is discarded.
  • the antler extract is concentrated and used, there is a problem in that the gelation occurs when the solid content is 20% or more.
  • a pill type there is an advantage of taking antler itself, but there is a problem that it is difficult to completely absorb the active ingredient in the body.
  • Korean Laid-Open Patent Publication No. 10-2003-0073134 describes a method of extracting antler which is powdered by adding water to fine antler, heating and extracting it under reduced pressure, and then concentrating and spray-drying the filtrate.
  • the extraction solution is powdered to improve the extraction method and ease of taking, the conventional problem is that a significant amount of the active ingredient contained in the extraction foil is not used and is discarded.
  • Korean Laid-Open Patent Publication No. 10-2005-0090041 describes a method of extracting antler, which is followed by hot water extraction, proteolytic enzyme treatment, and hydrochloric acid decomposition after pulverizing antler or rust.
  • the process proceeds to several stages, which is complicated and time-consuming, and there is a problem in that the use of strong acid destroys the bioactive substances useful for the human body.
  • the conventional antler extract thus obtained has an unpleasant odor and color and is difficult to apply to foods and cosmetics, and only a very small amount is used as a raw material.
  • the present invention is to solve the problems of the conventional antler extraction method, in the present invention, by using only two fermentation methods at low temperature so as not to destroy the physiologically active substance unique to antler, inducing a smooth extraction sufficient short-term decomposition It is an object to provide a method for producing a antler fermentation extract having a high content of the active ingredient to be made.
  • the present invention aims to provide an extract and a complex obtained according to the production method of the present invention.
  • the present invention in order to increase the stability of the antler fermented extract obtained in this way by using a molecular encapsulation technology, designed in a stable formulation to light, air, heat, acid and functional health foods and cosmetics that can maintain anti-inflammatory activity even during the shelf life
  • the purpose is to provide.
  • step 1 Adding purified water to the sliced antler, followed by hot water extraction and filtration (step 1);
  • step 2 Adding a Bacillus strain culture solution to the liquid powder obtained in step 1 and fermenting to obtain a first fermentation extract (step 2); And
  • step 3 After adding purified water to the antler obtained in step 1, aspergillus strain culture solution is added and fermented to obtain a second fermentation extract (step 3). .
  • stirring and molecular encapsulation After adding a clathrate complex to at least one selected from the group consisting of the first fermentation extract, the second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract, stirring and molecular encapsulation It is preferable to further include.
  • step 1 200 to 500 parts by weight of purified water is added per 100 parts by weight of chopped antler, and hot water extraction is preferably performed at 80 to 100 ° C.
  • step 2 10 to 30 parts by weight of the Bacillus strain culture solution is added per 100 parts by weight of chopped deer antler, fermentation is preferably carried out for 2 to 3 days at 30 to 40 °C.
  • Bacillus strain culture solution contains Bacillus casei.
  • step 3 10 to 30 parts by weight of Aspergillus strain culture solution is added per 100 parts by weight of chopped deer antler, it is preferable that the fermentation is carried out for 2 to 3 days at 50 to 70 °C.
  • the Aspergillus strain culture contains aspergillus casei .
  • At least one selected from the group consisting of a first fermentation extract, a second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract contains 20 to 30 parts by weight of cyclodextrin per 100 parts by weight of this mixture.
  • the clathrate complex is selected from cyclodextrins, crown ethers, polyoxyalkylenes, proporines, polysiloxanes, popazenes, zeolites.
  • the present invention also provides a complex formed from the fermentation extract and the inclusion complex obtained by adding and fermenting a Bacillus strain culture medium to the liquid powder obtained by hot water extraction of the cut antler.
  • the present invention also provides a complex formed from the fermentation extract and the inclusion complex obtained by adding and fermenting the Aspergillus strain culture solution to the solid antler obtained by hot water extraction of the cut antler.
  • the present invention is added to the fermentation broth obtained by adding and fermenting Bacillus strain culture liquid to the liquid powder obtained by hot water extraction of the sliced antler, and the Aspergillus strain culture solution to the solid antler obtained by hot water extraction of the sliced antler; A mixture of fermentation extracts obtained by fermentation and a complex formed from the inclusion complexes are provided.
  • the clathrate complex is selected from cyclodextrins, crown ethers, polyoxyalkylenes, proporines, polysiloxanes, popazenes, zeolites.
  • the present invention also provides a liquid antler fermentation extract of liquid form obtained by autoclaving the product prepared according to the production method of the present invention, or the complex of the present invention.
  • the present invention also provides a powdered antler fermentation extract obtained by autoclaving and lyophilizing the product prepared according to the production method of the present invention, or the complex of the present invention.
  • the present invention is a functional health food containing at least one selected from the antler fermentation extract prepared according to the production method of the present invention, the complex of the present invention, and liquid and powdered antler fermentation extract as an active ingredient, and Provide functional cosmetics.
  • the present invention can solve the problem of the destruction of physiologically active substances by conventional hydrothermal extraction, strong acid, hydrolysis using enzyme by extracting enough active ingredient of antler in a short time by fermentation extraction method using Bacillus and Aspergillus strain only. In addition, it is possible to obtain a high stability of the antler fermentation extract with a high content of a bioactive substance by the molecular encapsulation method.
  • 1 is a photograph showing the molecular weight size of the antler according to fermentation time.
  • Figure 2 is a graph showing the thermal stability test of the gangliosides for each extraction method.
  • 3 is a graph illustrating acid stability tests of gangliosides according to extraction methods.
  • Figure 4 is a graph showing the effect of fermented antler molecular capsules on IL-6 production in serum after simultaneous LPS treatment.
  • 5 is a graph showing the effect of fermented antler molecular capsules on MCP-1 production in serum after simultaneous LPS treatment.
  • FIG. 6 is a graph showing the effect of fermented antler molecular capsules on IL-1b production in serum after simultaneous LPS treatment.
  • FIG. 7 is a graph showing the effect of fermented antler molecular capsules on TNF-a production in serum after simultaneous LPS treatment.
  • a first aspect of the present invention provides a method for producing a antler fermentation extract from antler. More specifically, the present invention comprises the steps of adding purified water to the fine antler and hot water extraction and filtration (step 1); Adding a Bacillus strain culture solution to the liquid powder obtained in step 1 and fermenting to obtain a first fermentation extract (step 2); And adding purified water to the antler obtained in step 1, followed by adding and fermenting the Aspergillus strain culture medium to obtain a second fermentation extract (step 3). do.
  • step 1 purified water is added to the sliced antler, followed by hot water extraction and filtration. More specifically, 200 to 500 parts by weight of purified water is added and more preferably 300 to 40 parts by weight based on 100 parts by weight of dried and shredded antler. This mixed solution is extracted at 80 ° C to 100 ° C for 2 to 5 hours and filtered. If the amount of purified water is less or more than the range described above, the extraction efficiency may be lowered or diluted, which may cause economic problems in subsequent fermentation steps.
  • the obtained product is divided into a liquid component and an antler component.
  • the antler component means solids other than the liquid component of the material obtained by hot water extraction, which means a solid component which contains a lot of keratin and is not dissolved as water.
  • step 2 the Bacillus strain culture solution is added to the liquid powder obtained in the above step and fermented to obtain a first fermentation extract. More particularly, in this step, 10 to 30 parts by weight of Bacillus strain culture medium, preferably 20 to 25 parts by weight, and 30 ° C. to 40 ° C., preferably 35 ° C., per 100 parts by weight of finely divided antler in the liquid powder obtained in the step. Fermentation is carried out for 2 to 3 days at -37 °C.
  • Bacillus strain culture medium is used, and the fermentation strains contained therein include Bacillus casei (Accession No. KACC 91310P ), B. subtilis , B. natto , B. natto , and Bacillus. B. brevis is appropriate.
  • Bacillus strain culture of the present invention is proteus peptone no. 3 310.0g, beef extract 10.0g, yeast extract 5.0g, dextrose 20.0g, polysorbate 80 1.0g, ammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate 0.05g, dipotassium phosphate
  • the solution was incubated overnight by inoculating 20 g of Lactobacillus casei (KACC 91310P).
  • the fermentation temperature is lower or higher than the above-described temperature range, the fermentation does not proceed or the strain loses activity so that the fermentation does not proceed.
  • filtration may be performed to remove unreacted solids.
  • Filtration is preferably performed at least once using a 0.1-3 ⁇ m filter. If the pore size of the filter is less than 0.1 ⁇ m, the filter is very precise and extremely expensive (ultrafiltration method), so there is a problem of low economic efficiency. If the pore size exceeds 3 ⁇ m, there is no bactericidal power and fungi or bacteria are included in the final extract and contaminated during distribution. There is a problem that is likely to be.
  • the first fermentation extract is obtained through the first fermentation extraction step of this step.
  • step 3 10-30 parts by weight, preferably 20-25 parts by weight of Aspergillus strain culture medium is added to the antler obtained in step 1, and then 50 ° C to 70 ° C, preferably 60 ° C to 65 ° C. Fermentation is carried out for 2 to 3 days at °C. Fermentation of antler buckwheat has many keratin components, so it is appropriate to ferment at a high temperature because perfect fermentation does not proceed at relatively low fermentation temperature conditions.
  • Aspergillus strain culture solution is used in this fermentation step, and the fermented strains contained therein are aspergillus casei , aspergillus ducki ( A. oryzae ), and aspergillus niger ( A. niger). ), A. glaucus , and aspergillus niger , among others.
  • the Aspergillus strain culture solution of the present invention can be prepared as follows: Aspergillus Niger KK2 strain 0.1 %% after incubating for 7 days at 28 °C by inoculating in a potato (potato dextrose agar) slope medium Spores were suspended by the addition of Tween 80 solution. 10 mL of the spore suspension (3.5 ⁇ 10 7 spores per 1.0 mL) was inoculated into a 250 mL Erlenmeyer flask containing 90 mL of malt extract and incubated for 48 hours at 200 rpm in a shaker at 28 ° C. seed culture).
  • a 250 mL Erlenmeyer flask containing 47.5 mL of the culture medium was sterilized at 121 ° C. and 1.5 atm for 20 minutes and then seeded. 2.5 mL of the inoculation was incubated for 5 days at 200 rpm in a shaker at 28 °C.
  • the culture medium contains 2.5% rice straw (0.3 mm or less), 1.25% wheat bran (0.4 mm or less), 4.5% corn steep liquor, 0.125% industrial seed yeast It consisted of industrial yeast extract, 0.65% potassium monobasic phosphate, 0.05% copper sulfate pentahydrate, 0.01% cobalt sulfate heptahydrate, and pH was adjusted to 7.0 with 5N sodium hydroxide.
  • filtration may be performed to remove unreacted solids.
  • Filtration is preferably performed at least once using a 0.1-3 ⁇ m filter. If the pore size of the filter is less than 0.1 ⁇ m, the filter is very precise and extremely expensive (ultrafiltration method), so there is a problem of low economic efficiency. If the pore size exceeds 3 ⁇ m, there is no bactericidal power and fungi or bacteria are included in the final extract and contaminated during distribution. There is a problem that is likely to be.
  • the method of the present invention further comprises adding a clathrate complex to the mixture of the first fermentation extract and / or the second fermentation extract, followed by stirring to obtain molecular encapsulation, in order to obtain the antler fermentation extract with increased stability of the present invention.
  • a clathrate complex to the mixture of the first fermentation extract and / or the second fermentation extract, followed by stirring to obtain molecular encapsulation, in order to obtain the antler fermentation extract with increased stability of the present invention. It includes. More specifically, 20 to 30 parts by weight of cyclodextrin is added to 100 parts by weight of the mixed solution of the first fermentation extract and / or the second fermentation extract and stirred at 60 ° C. to 70 ° C. for 4 hours to encapsulate the bioactive substance. In order to more stabilize its molecular capsule, it is cooled slowly to room temperature to prepare a highly stable capsule.
  • the inclusion complex is preferably cyclodextrin, crown ether, polyoxyalkylene, proporine, polysiloxane, popazene, or zeolite, with cyclodextrin being more preferred.
  • the extract or complex of the present invention prepared as described above includes a liquid obtained by performing autoclaving and a powder product lyophilized thereof.
  • autoclaving is carried out at 121 ° C. for 30 minutes.
  • the fermentation extract obtained in the present invention is extracted by two fermentation methods at low temperature, biodegradable substances are not destroyed, and the antler component contained in the extract has many advantages over the extract obtained by the conventional extraction method, and also the molecular encapsulation.
  • By increasing the stability by having the advantage of designing a stable formulation to light, air, heat, acid bar can be used as an active ingredient in functional health food and cosmetics.
  • the rusted mollusk separated in the above (1) was added to 60 kg of purified water, and 1 kg of aspergillus niger culture solution was added thereto, followed by 2 at 60 ° C. Fermentation was daily. Thereafter, filtration was performed using a 1 ⁇ m filter to remove unreacted solids.
  • the composition thus prepared was lyophilized and powdered.
  • the obtained lyophilized powder was able to obtain more than 33kg.
  • 1 kg of dried antler was crushed using a crusher. 1 kg of crushed antler powder was added to 5 L of distilled water, and extracted at 100 ° C. for 7 hours and filtered. At this time, the primary filtration was performed by a 2 ⁇ m filter, the secondary filtration by a 0.45 ⁇ m filter, and the tertiary filtration by a 0.2 ⁇ m filter.
  • Example 1 The total amount of nitrogen of the antler extract finally obtained in Example 1 and Comparative Examples 1 and 2 was analyzed. The results are shown in Table 1 below.
  • the fermented antler molecular capsule according to the present invention can be seen that the total nitrogen and amino acid content is increased several times to several tens of times compared to the antler extract (Comparative Examples 1 and 2) obtained by the conventional method.
  • BSA bovine serum albumin
  • the sample was dissolved in distilled water and the absorbance was measured at 530 nm by carbazole reaction using a UV spectrophotometer, and the content was determined by calculating the content for D-glucuronic acid lactone.
  • Absorbance was measured using a UV spectrophotometer at 530 nm by carbazole reaction using NZP chondroitin sulfate as a standard.
  • the sample was dissolved in distilled water and the absorbance was measured using a UV spectrophotometer at 530 nm by carbazole reaction, and the content was determined by calculating the content for NZP chondroitin sulfate.
  • the absorbance was measured at 549 nm using a Warren method using a UV spectrophotometer, and the content was determined by calculating the content for N-acetylneuraminic acid.
  • Chondroitinase ABC (C2905, Sigma) from Proteus vulgaris was dissolved in 50 mM Tris-HCl 60 mM sodium acetate buffer (pH 8.0) to 1 U / ml.
  • NZP CS NZP CS as a standard, the standard and the sample were dissolved at 10 mg / ml, mixed with a buffer, 30 mU of enzyme, and reacted at 37 ° C. for 12 hours to analyze the content of chondroitin sulfate.
  • EGF-specific ELISA For the analysis of EGF, human EGF-specific ELISA was performed on antler hydrothermal extract, hydrolyzate and fermented extract. In order to prepare a standard curve, the concentration range having linearity was selected through linear regression analysis in the range of 250 pg / ml ⁇ 0 pg / ml, and absorbance analysis was performed by applying three samples to the test in this section. From this, the EGF of the sample was analyzed according to the concentration of the standard EGF.
  • a calibration curve was prepared from peak integration values of Rf 0.39 and 0.42 obtained by dissolving the ganglioside standard (Sigma Co.) in methanol, developing by TLC method, and scanning at a wavelength of 550 nm with a zig-zag TLC scanner.
  • each sample was performed in the same manner as in the standard method to quantify the gangliosides of each sample from the peak integration values of Rf 0.39 and 0.42.
  • the hydrothermal extract, hydrolyzed antler extract, and extracted by fermentation and molecular encapsulation can be seen that the bioactive substance contains a large amount.
  • Example 1 Comparative Example 2 and Example 1 were prepared by the fermentation extraction method without the molecular capsules 4 at 80 °C Changes in gangliosides were measured under severe conditions of temperature storage for one week and acidic conditions for four weeks at pH 1. The results are shown in FIGS. 2 and 3.
  • the antler extracted from hot water at a high temperature shows a significant decrease in the content of gangliosides over time, and in the case of fermentation with a relatively low extraction temperature, the ganglioside content is higher than that of hot water extraction, but this also decreases.
  • the ganglioside content of the molecular encapsulated deer antler did not change much as the initial state over time.
  • the antler extracted from hot water at high temperature shows a significant decrease in the content of gangliosides over time in strong acid conditions, and in the case of fermentation with a relatively low extraction temperature, the ganglioside content is higher than that of hot water extraction. It can be seen that the decrease. However, the content of the gangliosides of the antler molecule encapsulated in the molecular weight did not change much like the initial state over time.
  • mice were used in the experiment after adapting the Balb / c mice purchased from the Korea Experimental Animal Center in the laboratory environment for one week.
  • the conditions of the animal kennel were a conventional system, 22 ⁇ 2 ° C., 12 hours of the day were illuminated at 200-300 Lux, and 12 hours were all blocked.
  • Animals were supplied with solid feed (more than 22.1% crude protein, less than 8.0% crude fat, less than 5.0% crude fiber, less than 8.0% crude ash, more than 0.6% calcium, more than 0.4% phosphorus, Samyangsa, no antibiotics) and water.
  • lipopolysaccharide is manufactured by Sigma (Sigma Co., USA), physiological saline is a foreign pharmaceutical product, and IL-1 ⁇ , IL-6, TNF- ⁇ , and MCP-1 ELISA kit Millipore Inc. (USA) was used, and other reagents were used as express reagents.
  • the instrument used in this experiment was centrifuged. (Beckman Co., USA), roller mixing. (Gowon scientific technology Co., Korea), vortex mixer (Vision scientific Co., Korea), Luminex (Millipore, Co., USA) and the like were used.
  • LPS lipopolysaccharide
  • IL-6 was measured in the LPS-induced acute inflammation model after administration of BALB / c mice for 7 days after fermentation antler capsules.
  • fermented antler molecular capsule 100 administration group showed 301.6 ⁇ 56.7 (pg / ml)
  • fermented antler molecular capsule 200 administration group showed 212.2 ⁇ 9.8 (pg / ml), significantly increased compared to normal group.
  • the concentration-dependency was significantly higher than that of the control group. (** p ⁇ 0.01, *** p ⁇ 0.001) decreased.
  • the results are shown in FIG.
  • IL-1b was measured in LPS-induced acute inflammation model after administration of fermented antler molecular capsules to BALB / c mice for 7 days, and 25.6 ⁇ 5.6 (pg / ml) in control group and 116.8 ⁇ in control group 8.6 (pg / ml), 61.2 ⁇ 11.1 (pg / ml) of fermented antler molecular capsule 100 and 51.6 ⁇ 9.2 (pg / ml) of fermented antler molecular capsule 200 were significantly increased compared to normal group. In the fermented deer antler capsule group, the concentration-dependence was significantly higher than that of the control group. (** p ⁇ 0.01, *** p ⁇ 0.001) decreased. The results are shown in FIG.
  • the present invention can extract the antler by the most natural fermentation method and reduce the destruction of physiologically active substances of the antler, increase the content of the active ingredient and also increase the productivity, it is usefully used as a manufacturing method of the antler extract used in various fields Can be.
  • the present invention is suitable for wide use in food, cosmetics, etc. by systematically revealing the anti-inflammatory activity.

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Abstract

The present invention relates to a method for preparing a fermented deer antler extract having an anti-inflammatory activity, an extract obtained by the method and to the use of such extract. The method comprises :a step (step 1) of adding purified water to chopped deer antler and performing hot water extraction and filtering on the resultant material; a step (step 2) of adding a culture solution for Bacillus sp. strain to the liquid content obtained in step 1 and fermenting the resultant material to obtain a first fermented liquid extract; and a step (step 3) of adding purified water to the solid content of deer antler obtained in the first step, adding a culture solution for Aspergiullus sp. strain to the resultant material and fermenting the resultant material to obtain a second fermented liquid extract. According to the present invention, deer antler extracts can be prepared by the most environmentally-friendly fermentation, destruction of physiological active substance of deer antler is reduced while increasing the content of active ingredient, and productivity can be also improved. Therefore, the method of the present invention can be valuably used as a method for preparing deer antler extracts which may be used in various fields. Particularly, the method of the present invention systematically discloses an anti-inflammatory activity, and therefore, can be suitably and widely used in foods, cosmetics, etc.

Description

항염 활성을 갖는 발효녹용 추출물의 제조 방법, 이로부터 얻어진 추출물 및 이러한 추출물의 용도 Method for preparing fermented antler extract having anti-inflammatory activity, extract obtained therefrom and use of such extract
본 발명은 녹용 추출물을 제조하는 방법, 이로부터 얻어진 추출물 및 이러한 추출물의 용도에 관한 것으로서, 보다 특히 발효 추출을 이용하여 녹용 추출물을 제조하는 방법, 이로부터 얻어진 추출물 및 이러한 추출물의 용도에 관한 것이다. The present invention relates to a method for preparing an antler extract, an extract obtained therefrom and the use of such an extract, and more particularly to a method for producing an antler extract using fermented extract, an extract obtained therefrom and the use of such an extract.
녹용(Cervi Parvum Corun)은 보통 사슴의 뿔을 말하며, 구체적으로 자라기 시작한지 2개월 이내의 아직 각질화가 되지 않아 만져보면 약간 물렁할 정도로 조직이 연하고 털이 골고루 덮여 있는 수컷의 뿔을 말한다. 가을이 되면 물렁거리던 뿔이 단단하게 각질화가 되는데, 이는 발정기에 암컷을 차지하기 위한 싸움을 위한 것으로, 이렇게 각질화된 뿔이 바로 녹용에 비해 효능이 떨어지는 녹각이다. 이 녹각이 채취시기를 놓쳐 칼슘화된 후 단단해져서 저절로 떨어진 것을 낙각이라 한다.Deer antler (Cervi Parvum Corun) usually refers to the horns of a deer. Specifically, it refers to a male's horn that is slightly soft and covered with hairs. In the fall, the horns that have been softened are keratinized, which is for the fight to occupy females during the estrus, and these keratinized horns are less effective than antlers. This greenery misses the harvest time, becomes calcium, hardens, and then falls on its own.
동의보감 등의 문헌에도 녹용은 강장작용, 보혈작용, 강정작용, 진통작용, 조혈작용, 생장발육촉진작용, 심부전증 치료작용 및 기능항진작용 등이 있는 것으로 기록되어 있으며, 이밖에도 피로회복, 신체활력증강 및 신장의 이뇨기능강화효과 등 많은 효능을 갖고 있는 것으로 알려져 있다.In the literature such as Dongbobom, antler is reported to have tonic, blood, jeongjeong, analgesic, hematopoietic, growth-promoting action, heart failure treatment and hyperactivity, etc. In addition, fatigue recovery, physical vitality improvement and It is known to have a lot of efficacy, such as strengthening diuretic function of the kidney.
전통한방에서 사용하는 녹용의 복용방법으로는, 녹용을 여러 종류의 생약제들과 혼합, 분쇄한 후 물을 용매로 하여 가열 추출하여 그 여액을 복용하는 방법과, 생약제들을 분쇄하여 가루로 만든 뒤 환(丸)으로 해서 복용하는 방법이 주로 이용되고 있다. 위의 두 가지 방법은 각기 장단점이 있는데, 추출하여 복용하는 것은 녹용의 유효성분을 흡수하기 쉬운 반면에 추출박에 포함되어 있는 유효성분은 버리게 된다. 그리고 녹용을 추출한 후 농축하여 사용할 경우에는 고형분 함량 20% 이상의 경우에 겔화되는 문제점이 있어 제품에 응용하기 어려운 문제가 있다. 환제형의 경우에는 녹용 자체를 섭취한다는 이점이 있으나 체내에서 유효성분의 완전한 흡수가 어렵다는 문제점이 있다. As a method of taking antler used in traditional Chinese medicine, antler is mixed with various kinds of herbal medicines, pulverized, heated and extracted with water as a solvent to take the filtrate, and the herbal medicines are pulverized and powdered. (丸) How to take is mainly used. The above two methods have advantages and disadvantages. The extraction and taking are easy to absorb the active ingredient of the antler, while the active ingredient contained in the extract is discarded. In addition, when the antler extract is concentrated and used, there is a problem in that the gelation occurs when the solid content is 20% or more. In the case of a pill type, there is an advantage of taking antler itself, but there is a problem that it is difficult to completely absorb the active ingredient in the body.
한국공개특허 제10-2003-0073134호에서는 세절한 녹용에 물을 가하고 가열, 감압 추출한 후 여액을 농축, 분무 건조시켜 분말화하는 녹용 추출 방법을 기술하고 있는데, 이 방법은 전통한방에서 사용하는 열수 추출법을 일부 개선하고 복용이 용이하도록 추출액을 분말화하고 있으나, 추출박에 포함된 상당량의 유효성분이 이용되지 못하고 폐기되는 종래의 문제점을 그대로 가지고 있다. 또, 한국공개특허 제10-2005-0090041호에서는 녹용 또는 녹각을 분쇄한 후 온수추출, 단백분해효소 처리 및 염산 분해 처리를 하는 녹용 추출 방법을 기술하고 있는데, 이 방법에서는 온수추출의 한계를 극복하기 위하여 추가로 단백분해효소 처리 및 염산 분해 처리를 실시하고 있는데, 공정이 여러 단계로 진행되어 복잡하고 시간이 많이 소요되며, 강산의 사용으로 인체에 유용한 생리활성 물질들이 파괴되는 문제점이 있다. 또, 이렇게 얻어진 종래의 녹용 추출물은 대부분 불쾌한 냄새 및 색도를 갖고 있어 식품이나 화장품에 적용하는데 어려움이 있고, 원료로서 극히 미량만이 사용되고 있는 실정이다. Korean Laid-Open Patent Publication No. 10-2003-0073134 describes a method of extracting antler which is powdered by adding water to fine antler, heating and extracting it under reduced pressure, and then concentrating and spray-drying the filtrate. Although the extraction solution is powdered to improve the extraction method and ease of taking, the conventional problem is that a significant amount of the active ingredient contained in the extraction foil is not used and is discarded. In addition, Korean Laid-Open Patent Publication No. 10-2005-0090041 describes a method of extracting antler, which is followed by hot water extraction, proteolytic enzyme treatment, and hydrochloric acid decomposition after pulverizing antler or rust. In order to further perform protease treatment and hydrochloric acid decomposition treatment, the process proceeds to several stages, which is complicated and time-consuming, and there is a problem in that the use of strong acid destroys the bioactive substances useful for the human body. In addition, the conventional antler extract thus obtained has an unpleasant odor and color and is difficult to apply to foods and cosmetics, and only a very small amount is used as a raw material.
본 발명은 이러한 종래의 녹용 추출 방법의 문제점을 해결하고자 하는 것으로, 본 발명에서는 녹용 고유의 생리활성 물질이 파괴되지 않도록 저온에서 2차례의 발효방법만을 사용하면서도 원활한 추출을 유도하여 단시간 내에 충분한 분해가 이루어지도록 함으로써 유효성분의 함량이 높은 녹용 발효 추출물을 제조하는 방법을 제공하는 것을 목적으로 한다.The present invention is to solve the problems of the conventional antler extraction method, in the present invention, by using only two fermentation methods at low temperature so as not to destroy the physiologically active substance unique to antler, inducing a smooth extraction sufficient short-term decomposition It is an object to provide a method for producing a antler fermentation extract having a high content of the active ingredient to be made.
또한, 본 발명에서는 본 발명의 제조방법에 따라 얻어진 추출물 및 복합체를 제공하는 것을 목적으로 한다.In addition, the present invention aims to provide an extract and a complex obtained according to the production method of the present invention.
또한, 본 발명에서는 이렇게 얻어진 녹용 발효추출물의 안정성을 증대시키기 위하여 분자캡슐화 기술을 이용하여 빛, 공기, 열, 산에 안정한 제형으로 설계하고 유통기간 내에서도 항염 활성을 유지 할 수 있는 기능성 건강식품 및 화장품을 제공하는 것을 목적으로 한다.In addition, in the present invention, in order to increase the stability of the antler fermented extract obtained in this way by using a molecular encapsulation technology, designed in a stable formulation to light, air, heat, acid and functional health foods and cosmetics that can maintain anti-inflammatory activity even during the shelf life The purpose is to provide.
상기와 같은 목적을 달성하기 위하여, 본 발명은In order to achieve the above object, the present invention
세절된 녹용에 정제수를 첨가하고 열수 추출 및 여과하는 단계(단계 1); Adding purified water to the sliced antler, followed by hot water extraction and filtration (step 1);
단계 1에서 얻어진 액상분에 바실러스 균주 배양액을 첨가하고 발효시켜 제 1 발효 추출액을 수득하는 단계(단계 2); 및 Adding a Bacillus strain culture solution to the liquid powder obtained in step 1 and fermenting to obtain a first fermentation extract (step 2); And
단계 1에서 얻어진 녹용박에 정제수를 첨가한 후에 아스퍼질러스 균주 배양액을 첨가하고 발효시켜 제 2 발효 추출액을 수득하는 단계(단계 3)를 포함하는, 녹용으로부터 녹용 발효 추출물을 제조하는 방법을 제공한다.After adding purified water to the antler obtained in step 1, aspergillus strain culture solution is added and fermented to obtain a second fermentation extract (step 3). .
본 발명에 따를 경우, 상기 제 1 발효 추출액, 제 2 발효 추출액, 및 제 1 발효 추출액 및 제 2 발효 추출액의 혼합물로 이루어진 군으로부터 선택된 하나 이상에 포접 복합체를 첨가한 후에, 교반하여 분자캡슐화시키는 단계를 추가로 포함하는 것이 바람직하다.According to the present invention, after adding a clathrate complex to at least one selected from the group consisting of the first fermentation extract, the second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract, stirring and molecular encapsulation It is preferable to further include.
본 발명에 따를 경우, 상기 단계 1에서, 세절된 녹용 100 중량부 당 200 내지 500 중량부의 정제수가 첨가되며, 열수 추출이 80 내지 100℃에서 수행되는 것이 바람직하다.According to the present invention, in step 1, 200 to 500 parts by weight of purified water is added per 100 parts by weight of chopped antler, and hot water extraction is preferably performed at 80 to 100 ° C.
본 발명에 따를 경우, 상기 단계 2에서, 세절된 녹용 100 중량부 당 10 내지 30 중량부의 바실러스 균주 배양액이 첨가되며, 발효가 30 내지 40℃에서 2 내지 3일 동안 수행되는 것이 바람직하다.According to the present invention, in step 2, 10 to 30 parts by weight of the Bacillus strain culture solution is added per 100 parts by weight of chopped deer antler, fermentation is preferably carried out for 2 to 3 days at 30 to 40 ℃.
본 발명에 따를 경우, 바실러스 균주 배양액이 바실러스 카제이(Bacillus casei)를 함유하는 것이 바람직하다.According to the present invention, it is preferred that the Bacillus strain culture solution contains Bacillus casei.
본 발명에 따를 경우, 상기 단계 3에서, 세절된 녹용 100 중량부 당 10 내지 30 중량부의 아스퍼질러스 균주 배양액이 첨가되며, 발효가 50 내지 70℃에서 2 내지 3일 동안 수행되는 것이 바람직하다.According to the present invention, in step 3, 10 to 30 parts by weight of Aspergillus strain culture solution is added per 100 parts by weight of chopped deer antler, it is preferable that the fermentation is carried out for 2 to 3 days at 50 to 70 ℃.
본 발명에 따를 경우, 아스퍼질러스 균주 배양액이 아스퍼질러스 카제이(aspergillus casei)를 함유하는 것이 바람직하다.According to the present invention, it is preferred that the Aspergillus strain culture contains aspergillus casei .
본 발명에 따를 경우, 제 1 발효 추출액, 제 2 발효 추출액, 및 제 1 발효 추출액 및 제 2 발효 추출액의 혼합물로 이루어진 군으로부터 선택된 하나 이상에 이러한 혼합물 100 중량부 당 20 내지 30 중량부의 사이클로덱스트린을 첨가한 후에, 60 내지 70℃에서 교반하고, 이를 상온까지 서서히 냉각시켜 분자캡슐화 시키는 것이 바람직하다.According to the present invention, at least one selected from the group consisting of a first fermentation extract, a second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract contains 20 to 30 parts by weight of cyclodextrin per 100 parts by weight of this mixture. After addition, it is preferable to stir at 60 to 70 ℃, it is gradually cooled to room temperature to molecular encapsulation.
본 발명에 따를 경우, 포접 복합체가 사이클로덱스트린, 크라운 에테르, 폴리옥시알킬렌, 프로포린, 폴리실록산, 포파젠, 제올라이트로부터 선택된 것이 바람직하다.According to the invention, it is preferred that the clathrate complex is selected from cyclodextrins, crown ethers, polyoxyalkylenes, proporines, polysiloxanes, popazenes, zeolites.
또한, 본 발명은 세절된 녹용을 열수 추출하여 얻어진 액상분에 바실러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과 포접 복합체로부터 형성된 복합체를 제공한다.The present invention also provides a complex formed from the fermentation extract and the inclusion complex obtained by adding and fermenting a Bacillus strain culture medium to the liquid powder obtained by hot water extraction of the cut antler.
또한, 본 발명은 세절된 녹용을 열수 추출하여 얻어진 고형의 녹용분에 아스퍼질러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과 포접 복합체로부터 형성된 복합체를 제공한다.The present invention also provides a complex formed from the fermentation extract and the inclusion complex obtained by adding and fermenting the Aspergillus strain culture solution to the solid antler obtained by hot water extraction of the cut antler.
또한, 본 발명은 세절된 녹용을 열수 추출하여 얻어진 액상분에 바실러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과, 세절된 녹용을 열수 추출하여 얻어진 고형의 녹용분에 아스퍼질러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액의 혼합물, 및 포접 복합체로부터 형성된 복합체를 제공한다.In addition, the present invention is added to the fermentation broth obtained by adding and fermenting Bacillus strain culture liquid to the liquid powder obtained by hot water extraction of the sliced antler, and the Aspergillus strain culture solution to the solid antler obtained by hot water extraction of the sliced antler; A mixture of fermentation extracts obtained by fermentation and a complex formed from the inclusion complexes are provided.
본 발명에 따를 경우, 포접 복합체가 사이클로덱스트린, 크라운 에테르, 폴리옥시알킬렌, 프로포린, 폴리실록산, 포파젠, 제올라이트로부터 선택된 것이 바람직하다.According to the invention, it is preferred that the clathrate complex is selected from cyclodextrins, crown ethers, polyoxyalkylenes, proporines, polysiloxanes, popazenes, zeolites.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 생성물, 또는 본 발명의 복합체를 고압 멸균시켜 얻어진 액상형의 녹용 발효 추출물을 제공한다.The present invention also provides a liquid antler fermentation extract of liquid form obtained by autoclaving the product prepared according to the production method of the present invention, or the complex of the present invention.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 생성물, 또는 본 발명의 복합체를 고압 멸균시키고 이를 동결건조시켜 얻어진 분말형의 녹용 발효 추출물을 제공한다.The present invention also provides a powdered antler fermentation extract obtained by autoclaving and lyophilizing the product prepared according to the production method of the present invention, or the complex of the present invention.
또한, 본 발명은 본 발명의 제조방법에 따라 제조된 녹용 발효 추출물, 본 발명의 복합체, 및 액상형 및 분말형의 녹용 발효 추출물로부터부 선택된 것 중 적어도 하나를 유효성분으로 함유하는 기능성 건강식품, 및 기능성 화장품을 제공한다.In addition, the present invention is a functional health food containing at least one selected from the antler fermentation extract prepared according to the production method of the present invention, the complex of the present invention, and liquid and powdered antler fermentation extract as an active ingredient, and Provide functional cosmetics.
본 발명은 바실러스 및 아스퍼질러스 균주를 이용한 발효추출방법만으로 단시간 내에 녹용의 유효성분을 충분히 추출해 냄으로써 종래의 열수추출이나 강산, 효소를 이용한 가수분해법의 생리활성물질의 파괴에 대한 문제를 해소할 수 있으며, 분자캡슐화 방법에 의해 생리활성물질의 함량이 높은 녹용 발효추출물의 높은 안정성을 얻을 수 있다.The present invention can solve the problem of the destruction of physiologically active substances by conventional hydrothermal extraction, strong acid, hydrolysis using enzyme by extracting enough active ingredient of antler in a short time by fermentation extraction method using Bacillus and Aspergillus strain only. In addition, it is possible to obtain a high stability of the antler fermentation extract with a high content of a bioactive substance by the molecular encapsulation method.
도 1은 발효 시간에 따른 녹용의 분자량 크기를 도시한 사진이다.1 is a photograph showing the molecular weight size of the antler according to fermentation time.
도 2는 추출방법별 강글리오사이드류의 열안정성 시험을 도시한 그래프이다.Figure 2 is a graph showing the thermal stability test of the gangliosides for each extraction method.
도 3은 추출방법별 강글리오사이드류의 산안정성 시험을 도시한 그래프이다.3 is a graph illustrating acid stability tests of gangliosides according to extraction methods.
도 4는 LPS 동시 처리 후 혈청에서 IL-6 생산에 대한 발효녹용 분자캡슐의 효과를 도시한 그래프이다.Figure 4 is a graph showing the effect of fermented antler molecular capsules on IL-6 production in serum after simultaneous LPS treatment.
도 5는 LPS 동시 처리 후 혈청에서 MCP-1 생산에 대한 발효녹용 분자캡슐의 효과를 도시한 그래프이다.5 is a graph showing the effect of fermented antler molecular capsules on MCP-1 production in serum after simultaneous LPS treatment.
도 6은 LPS 동시 처리 후 혈청에서 IL-1b 생산에 대한 발효녹용 분자캡슐의 효과를 도시한 그래프이다.6 is a graph showing the effect of fermented antler molecular capsules on IL-1b production in serum after simultaneous LPS treatment.
도 7은 LPS 동시 처리 후 혈청에서 TNF-a 생산에 대한 발효녹용 분자캡슐의 효과를 도시한 그래프이다.7 is a graph showing the effect of fermented antler molecular capsules on TNF-a production in serum after simultaneous LPS treatment.
본 발명의 제 1 양태는 녹용으로부터 녹용 발효 추출물을 제조하는 방법을 제공한다. 보다 특히, 본 발명은 세절된 녹용에 정제수를 첨가하고 열수 추출 및 여과하는 단계(단계 1); 단계 1에서 얻어진 액상분에 바실러스 균주 배양액을 첨가하고 발효시켜 제 1 발효 추출액을 수득하는 단계(단계 2); 및 단계 1에서 얻어진 녹용박에 정제수를 첨가한 후에 아스퍼질러스 균주 배양액을 첨가하고 발효시켜 제 2 발효 추출액을 수득하는 단계(단계 3)를 포함하는, 녹용으로부터 녹용 발효 추출물을 제조하는 방법을 제공한다.A first aspect of the present invention provides a method for producing a antler fermentation extract from antler. More specifically, the present invention comprises the steps of adding purified water to the fine antler and hot water extraction and filtration (step 1); Adding a Bacillus strain culture solution to the liquid powder obtained in step 1 and fermenting to obtain a first fermentation extract (step 2); And adding purified water to the antler obtained in step 1, followed by adding and fermenting the Aspergillus strain culture medium to obtain a second fermentation extract (step 3). do.
이하에서는 본 발명의 녹용 발효추출물의 제조 방법을 각 단계별로 상세히 설명한다. Hereinafter will be described in detail for each step the manufacturing method of the antler fermentation extract of the present invention.
열수 추출 단계Hydrothermal extraction stage
단계 1에서는 세절된 녹용에 정제수를 첨가하고 열수 추출 및 여과한다. 보다 구체적으로, 건조되고 세절된 녹용 100 중량부에 대하여 정제수 200 내지 500 중량부를 가하고 보다 바람직하게는 300 내지 40 중량부를 가한다. 이러한 혼합액을 80℃ 내지 100℃에서 2 내지 5 시간 추출시키고 여과한다. 정제수의 양이 상기 기재된 범위 보다 적거나 많은 경우에는 추출효율이 저하되거나 희석되는 경우가 있어 이후의 발효단계에서 경제적이지 못한 문제점이 발생할 수 있다.In step 1, purified water is added to the sliced antler, followed by hot water extraction and filtration. More specifically, 200 to 500 parts by weight of purified water is added and more preferably 300 to 40 parts by weight based on 100 parts by weight of dried and shredded antler. This mixed solution is extracted at 80 ° C to 100 ° C for 2 to 5 hours and filtered. If the amount of purified water is less or more than the range described above, the extraction efficiency may be lowered or diluted, which may cause economic problems in subsequent fermentation steps.
이에 따라, 얻어진 생성물은 액상분 및 녹용분으로 나뉘어진다. 여기서 녹용분이라 함은 열수 추출에 따라 얻어진 물질 중 액상분을 제외한 나머지 고형분을 의미하는 것으로서, 이는 각질을 많이 함유하여 물로서 용해되지 않는 고형분을 의미한다.As a result, the obtained product is divided into a liquid component and an antler component. Here, the antler component means solids other than the liquid component of the material obtained by hot water extraction, which means a solid component which contains a lot of keratin and is not dissolved as water.
1차 발효추출 단계1st fermentation extraction stage
단계 2에서는 상기 단계에서 얻어진 액상분에 바실러스 균주 배양액을 첨가하고 발효시켜 제 1 발효 추출액을 수득한다. 보다 특히, 본 단계에서는 상기 단계에서 얻어진 액상분에 세절된 녹용 100 중량부 당 바실러스 균주배양액 10 내지 30 중량부, 바람직하게는 20 내지 25 중량부를 첨가하고 30℃ 내지 40℃, 바람직하게는 35℃ 내지 37℃에서 2일 내지 3일 동안 발효시킨다. In step 2, the Bacillus strain culture solution is added to the liquid powder obtained in the above step and fermented to obtain a first fermentation extract. More particularly, in this step, 10 to 30 parts by weight of Bacillus strain culture medium, preferably 20 to 25 parts by weight, and 30 ° C. to 40 ° C., preferably 35 ° C., per 100 parts by weight of finely divided antler in the liquid powder obtained in the step. Fermentation is carried out for 2 to 3 days at -37 ℃.
본 발효추출 단계에서는 바실러스 균주 배양액을 사용하는데, 이에 함유되는 발효균주로는 바실러스 카제이(Bacillus casei, 수탁번호 KACC 91310P ), 바실러스 서브틸리스(B. subtilis), 바실러스 나토(B. natto), 바실러스 브레비스(B. brevis)가 적절하다.In the fermentation extraction step, Bacillus strain culture medium is used, and the fermentation strains contained therein include Bacillus casei (Accession No. KACC 91310P ), B. subtilis , B. natto , B. natto , and Bacillus. B. brevis is appropriate.
일 예로서, 본 발명의 바실러스 균주 배양액은 프로테오스펩톤 no. 3 310.0g, 비프추출물 10.0g, 효모추출물 5.0g, 덱스트로스 20.0g, 폴리소베이트80 1.0g, 암모니늄시트레이트 2.0g, 소듐아세테이트 5.0g, 마그네슘설페이트 0.1g, 망간설페이트 0.05g, 디포타슘포스페이트 20g에 락토바실러스 카세이(KACC 91310P)를 접종하여 하룻밤 동안 배양한 액이다.As an example, Bacillus strain culture of the present invention is proteus peptone no. 3 310.0g, beef extract 10.0g, yeast extract 5.0g, dextrose 20.0g, polysorbate 80 1.0g, ammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate 0.05g, dipotassium phosphate The solution was incubated overnight by inoculating 20 g of Lactobacillus casei (KACC 91310P).
본 1차 발효추출 단계에서 발효온도가 상기 기술된 온도 범위 보다 낮거나 높은 경우에는 발효가 진행되지 않거나 균주가 활성을 잃어버려 발효가 진행되지 않게 된다.In the first fermentation extraction step, if the fermentation temperature is lower or higher than the above-described temperature range, the fermentation does not proceed or the strain loses activity so that the fermentation does not proceed.
1차 발효추출 단계를 거친 후 미반응 고형분을 제거하기 위한 여과를 실시할 수 있다. 여과는 0.1 내지 3 ㎛ 필터를 사용하여 1회 이상 여과를 수행하는 것이 바람직하다. 필터의 기공 크기가 0.1 ㎛ 미만인 경우에는 필터가 초정밀하여 초고가이므로(한외여과법) 경제성이 떨어지는 문제가 있고, 3 ㎛ 를 초과하는 경우는 제균력이 없어 진균이나 세균이 최종 추출물에 포함되어 유통 중 오염될 가능성이 높다는 문제가 있다. After the first fermentation extraction step, filtration may be performed to remove unreacted solids. Filtration is preferably performed at least once using a 0.1-3 μm filter. If the pore size of the filter is less than 0.1 μm, the filter is very precise and extremely expensive (ultrafiltration method), so there is a problem of low economic efficiency. If the pore size exceeds 3 μm, there is no bactericidal power and fungi or bacteria are included in the final extract and contaminated during distribution. There is a problem that is likely to be.
이에 따라, 본 단계의 1차 발효추출 단계를 거쳐 제 1 발효 추출액을 수득한다.Accordingly, the first fermentation extract is obtained through the first fermentation extraction step of this step.
2차 발효추출 단계Second fermentation extraction stage
단계 3에서는 상기 단계 1에서 얻어진 녹용박에 정제수를 첨가한 후에 아스퍼질러스 균주 배양액 10 내지 30 중량부, 바람직하게는 20 내지 25 중량부를 가하고, 50℃ 내지 70℃, 바람직하게는 60℃ 내지 65℃에서 2일 내지 3일 발효시킨다. 녹용박의 발효는 각질성분이 많아 상대적으로 낮은 발효온도 조건에서는 완벽한 발효가 진행되지 않기 때문에 높은 온도에서 발효하는 것이 적절하다.In step 3, 10-30 parts by weight, preferably 20-25 parts by weight of Aspergillus strain culture medium is added to the antler obtained in step 1, and then 50 ° C to 70 ° C, preferably 60 ° C to 65 ° C. Fermentation is carried out for 2 to 3 days at ℃. Fermentation of antler buckwheat has many keratin components, so it is appropriate to ferment at a high temperature because perfect fermentation does not proceed at relatively low fermentation temperature conditions.
본 발효추출 단계에서는 아스퍼질러스 균주 배양액을 사용하는데, 이에 함유되는 발효균주로는 아스퍼질러스 카제이(aspergillus casei), 아스퍼질러스 오리재(A. oryzae), 아스퍼질러스 나이거(A. niger), 아스퍼질러스 글라우쿠스(A. glaucus), 그중에서도 아스퍼질러스 나이거(aspergillus niger)가 적절하다.Aspergillus strain culture solution is used in this fermentation step, and the fermented strains contained therein are aspergillus casei , aspergillus ducki ( A. oryzae ), and aspergillus niger ( A. niger). ), A. glaucus , and aspergillus niger , among others.
일 예로서, 본 발명의 아스퍼질러스 균주 배양액은 하기와 같이 제조될 수 있다: 아스퍼질러스 나이거 KK2 균주를 PDA(potato dextrose agar) 사면배지에 접종하여 28℃에서 7일간 배양한 후 0.1% Tween 80 용액을 첨가하여 포자를 현탁시켰다. 포자현탁액(1.0 mL 당 3.5 × 10 7 포자) 10 mL를 90 mL의 맥아추출물(malt extract)이 들어있는 250 mL의 삼각플라스크에 접종한 후 28℃의 진탕배양기에서 200 rpm으로 48시간 종균 배양(seed culture) 하였다. 본 배양 배지 47.5 mL가 들어있는 250 mL의 삼각플라스크를 121℃, 1.5 기압에서 20분간 멸균한 후 종균 하였다. 2.5 mL을 접종하여 28℃의 진탕 배양기에서 200 rpm으로 5일간 배양하였다. 본 배양배지는 2.5%의 볏짚(rice straw, 0.3 mm 이하 크기), 1.25%의 밀기울(wheat bran, 0.4 mm 이하 크기), 4.5%의 옥수수 농침액(corn steep liquor), 0.125%의 산업종균 효모추출액(industrial yeast extract), 0.65%의 일염기 인산칼륨, 0.05%의 황산동 5수화물, 0.01%의 황산코발트 7수화물로 구성되었으며, 5N 수산화나트륨으로 pH를 7.0으로 조정되었다.As an example, the Aspergillus strain culture solution of the present invention can be prepared as follows: Aspergillus Niger KK2 strain 0.1 %% after incubating for 7 days at 28 ℃ by inoculating in a potato (potato dextrose agar) slope medium Spores were suspended by the addition of Tween 80 solution. 10 mL of the spore suspension (3.5 × 10 7 spores per 1.0 mL) was inoculated into a 250 mL Erlenmeyer flask containing 90 mL of malt extract and incubated for 48 hours at 200 rpm in a shaker at 28 ° C. seed culture). A 250 mL Erlenmeyer flask containing 47.5 mL of the culture medium was sterilized at 121 ° C. and 1.5 atm for 20 minutes and then seeded. 2.5 mL of the inoculation was incubated for 5 days at 200 rpm in a shaker at 28 ℃. The culture medium contains 2.5% rice straw (0.3 mm or less), 1.25% wheat bran (0.4 mm or less), 4.5% corn steep liquor, 0.125% industrial seed yeast It consisted of industrial yeast extract, 0.65% potassium monobasic phosphate, 0.05% copper sulfate pentahydrate, 0.01% cobalt sulfate heptahydrate, and pH was adjusted to 7.0 with 5N sodium hydroxide.
2차 발효추출 단계를 거친 후 미반응 고형물질을 제거하기 위한 여과를 실시할 수 있다. 여과는 0.1 내지 3 ㎛ 필터를 사용하여 1회 이상 여과를 수행하는 것이 바람직하다. 필터의 기공 크기가 0.1 ㎛ 미만인 경우에는 필터가 초정밀하여 초고가이므로(한외여과법) 경제성이 떨어지는 문제가 있고, 3 ㎛ 를 초과하는 경우는 제균력이 없어 진균이나 세균이 최종 추출물에 포함되어 유통 중 오염될 가능성이 높다는 문제가 있다. After the second fermentation extraction step, filtration may be performed to remove unreacted solids. Filtration is preferably performed at least once using a 0.1-3 μm filter. If the pore size of the filter is less than 0.1 μm, the filter is very precise and extremely expensive (ultrafiltration method), so there is a problem of low economic efficiency. If the pore size exceeds 3 μm, there is no bactericidal power and fungi or bacteria are included in the final extract and contaminated during distribution. There is a problem that is likely to be.
분자캡슐화 단계Molecular Encapsulation Step
본 발명의 제조방법은 본 발명의 안정성이 증대된 녹용 발효추출물을 얻기 위해, 상기 제 1 발효 추출액 및/또는 제 2 발효 추출액의 혼합물에 포접 복합체를 첨가한 후에, 교반하여 분자캡슐화시키는 단계를 추가로 포함한다. 보다 특히, 상기 제 1 발효 추출액 및/또는 제 2 발효 추출액의 혼합액 100 중량부에 사이클로덱스트린 20 내지 30 중량부를 투입하여 60℃ 내지 70℃에서 4시간 교반하여 생리활성물질을 분자캡슐화시킨다. 이의 분자캡슐을 보다 안정화하기 위하여 상온까지 서서히 냉각하여 안정성이 높은 캡슐을 제조한다. 포접 복합체에는 사이클로덱스트린, 크라운 에테르, 폴리옥시알킬렌, 프로포린, 폴리실록산, 포파젠, 또는 제올라이트가 바람직하며, 사이클로덱스트린이 더욱 바람직하다. 이에 따라, 제 1 녹용 발효 추출액 및 제 2 녹용 발효 추출액의 혼합물과 포접 복합체로부터 형성된 복합체를 형성시킨다.The method of the present invention further comprises adding a clathrate complex to the mixture of the first fermentation extract and / or the second fermentation extract, followed by stirring to obtain molecular encapsulation, in order to obtain the antler fermentation extract with increased stability of the present invention. It includes. More specifically, 20 to 30 parts by weight of cyclodextrin is added to 100 parts by weight of the mixed solution of the first fermentation extract and / or the second fermentation extract and stirred at 60 ° C. to 70 ° C. for 4 hours to encapsulate the bioactive substance. In order to more stabilize its molecular capsule, it is cooled slowly to room temperature to prepare a highly stable capsule. The inclusion complex is preferably cyclodextrin, crown ether, polyoxyalkylene, proporine, polysiloxane, popazene, or zeolite, with cyclodextrin being more preferred. Thereby, a complex formed from the inclusion complex with a mixture of the first antler fermentation extract and the second antler fermentation extract is formed.
이와 같이 제조된 본 발명의 추출물 또는 복합체는 고압멸균을 수행하여 얻어진 액상 및 이를 동결건조한 분말제품이 포함된다. 바람직하게, 고압멸균은 121℃에서 30분간 수행된다.The extract or complex of the present invention prepared as described above includes a liquid obtained by performing autoclaving and a powder product lyophilized thereof. Preferably, autoclaving is carried out at 121 ° C. for 30 minutes.
이에 따라 얻어진 제 1 발효 추출액 및 제 2 발효 추출액을 혼합한 녹용 추출물, 및/또는 이를 분자캡슐화하여 얻어진 복합체, 및/또는 액상형 또는 분말형의 녹용 발효 추출물은 유효 성분으로 하여 기능성 건강식품 및 화장품 등에 사용될 수 있다.The deer antler extract obtained by mixing the first fermentation extract and the second fermentation extract thus obtained, and / or the complex obtained by molecular encapsulation, and / or the liquid or powdered deer antler fermentation extract as an active ingredient as a functional health food and cosmetics Can be used.
본 발명에서 얻어진 발효 추출액은 저온에서 2차례의 발효방법에 의해 추출되는 바 생리활성 물질이 파괴되지 않고 추출액에 함유된 녹용 성분이 종래 추출방법에 의해 얻어진 추출액에 비해 많은 장점이 있으며, 또한 분자캡슐화에 의해 안정성을 증대시켜 빛, 공기, 열, 산에 안정한 제형을 설계할 수 있다는 장점을 가지고 있는 바, 기능성 건강식품 및 화장품의 유효성분으로 사용할 수 있다.The fermentation extract obtained in the present invention is extracted by two fermentation methods at low temperature, biodegradable substances are not destroyed, and the antler component contained in the extract has many advantages over the extract obtained by the conventional extraction method, and also the molecular encapsulation. By increasing the stability by having the advantage of designing a stable formulation to light, air, heat, acid bar can be used as an active ingredient in functional health food and cosmetics.
본 발명의 녹용 추출물 이외에 첨가될 건강식품 및 화장품의 부형제 및 기타 첨가제의 종류 및 양은 당업자에 의해 용이하게 선택될 수 있으며, 이에 대해서는 본 명세서에서 제한하지 않는다.Kinds and amounts of excipients and other additives of health foods and cosmetics to be added in addition to the antler extract of the present invention can be easily selected by those skilled in the art, which are not limited herein.
이하 구체적인 실시 예를 통해 본 발명을 보다 상세히 설명한다. 그러나, 하기 실시예에 의해 본 발명의 범위가 한정되는 것은 아니며, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 본 발명의 기술사상과 아래에 기재될 특허 청구범위의 균등범위 내에서 다양한 수정 및 변형이 가능한 것은 물론이다.Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the scope of the present invention is not limited by the following examples, and those of ordinary skill in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.
<실시예 1> <Example 1> 발효녹용 분자캡슐의 제조Preparation of Fermented Rust Molecular Capsule
(1) 건조되고 세절된 녹용 10kg을 정제수 35 ㎏에 첨가하고 90℃에서 4시간 추출시킨 후에 이를 여과를 통해 녹용박을 분리시켜 여액을 수득하였다.(1) 10 kg of dried and shredded antler was added to 35 kg of purified water, and extracted at 90 ° C. for 4 hours, and the antler was separated through filtration to obtain a filtrate.
(2) 1차 발효물을 얻기 위해, 수득된 여액에 바실러스 서브틸리스(bacillus subtilis) 배양액 1kg을 첨가한 후에 37℃에서 2일간 발효시켰다. 이후 1 ㎛ 필터를 사용하여 여과를 수행하여 미반응 고형분을 제거하였다.(2) In order to obtain a primary fermentation product, 1 kg of Bacillus subtilis culture solution was added to the obtained filtrate and then fermented at 37 ° C. for 2 days. Thereafter, filtration was performed using a 1 μm filter to remove unreacted solids.
(3) 2차 발효물을 얻기 위해, 상기 (1) 항에서 분리된 녹용박을 정제수 60 kg에 첨가하고, 여기에 아스퍼질러스 나이거(aspergillus niger) 배양액 1kg을 첨가한 후에 60℃에서 2일간 발효시켰다. 이후 1 ㎛ 필터를 사용하여 여과를 수행하여 미반응 고형분을 제거하였다.(3) In order to obtain a secondary fermented product, the rusted mollusk separated in the above (1) was added to 60 kg of purified water, and 1 kg of aspergillus niger culture solution was added thereto, followed by 2 at 60 ° C. Fermentation was daily. Thereafter, filtration was performed using a 1 μm filter to remove unreacted solids.
(4) 상기 발효 및 여과된 1, 2차 녹용 발효물을 혼합하고, 이 혼합액을 한천여과를 통하여 여과하였다. 이 여액에 사이클로덱스트린 25kg을 첨가하고 60℃에서 4시간 교반하여 생리활성물질을 분자캡슐화시켰다. 이의 분자캡슐을 보다 안정화하기 위하여 상온까지 서서히 냉각하여 안정성이 높은 캡슐을 제조하였다.(4) The fermented and filtered primary and secondary antler fermented products were mixed, and the mixed solution was filtered through agar filtration. 25 kg of cyclodextrin was added to the filtrate and stirred at 60 ° C. for 4 hours to encapsulate the bioactive substance. In order to stabilize its molecular capsules more slowly, it was cooled slowly to room temperature to prepare highly stable capsules.
이와 같이 제조된 조성물을 동결건조하여 분말화하였다. 이때 얻어진 동결건조된 분말은 33kg 이상을 얻을 수 있었다. The composition thus prepared was lyophilized and powdered. The obtained lyophilized powder was able to obtain more than 33kg.
이와 관련하여, 상기에서 얻어진 상기 1차, 2차 녹용 발효물의 분자량크기를 측정하기 위하여 전기영동실험을 수행하였다. 그 결과는 도 1에 나타내었다. 도 1로부터 발효가 진행될수록 분자량크기가 작아져 인체에 흡수가 용이한 크기로 전환됨을 알 수 있다.In this regard, electrophoretic experiments were performed to determine the molecular weight of the primary and secondary antler fermentation obtained above. The results are shown in FIG. As the fermentation proceeds from FIG. 1, it can be seen that the molecular weight is reduced to a size that is easily absorbed by the human body.
<비교예 1> Comparative Example 1 효소를 이용한 녹용 가수분해물의 제조Preparation of Antler Hydrolyzate Using Enzyme
건조된 녹용 1 ㎏을 파쇄기를 이용하여 파쇄하였다. 파쇄된 녹용분말 1 ㎏에 파파인(papain) 10 g 및 증류수 5 ㎏을 첨가한 후에 70℃에서 8시간 반응시켜 녹용분말을 가수분해시켰다. 가수분해된 용액을 여과시켜 미반응 고형분을 제거하였다. 이 때, 1차 여과는 2 ㎛ 필터로, 2차 여과는 0.45 ㎛ 필터로, 3차 여과는 0.2 ㎛ 필터로 수행하였다. 1 kg of dried antler was crushed using a crusher. 10 kg of papain and 5 kg of distilled water were added to 1 kg of the crushed antler powder, followed by reaction at 70 ° C. for 8 hours to hydrolyze the antler powder. The hydrolyzed solution was filtered to remove unreacted solids. At this time, the primary filtration was performed by a 2 μm filter, the secondary filtration by a 0.45 μm filter, and the tertiary filtration by a 0.2 μm filter.
<비교예 2> Comparative Example 2 열수추출을 이용한 녹용 추출물의 제조Preparation of Deer Antler Extract Using Hot Water Extraction
건조된 녹용 1 ㎏ 을 파쇄기를 이용하여 파쇄하였다. 파쇄된 녹용분말 1 ㎏을 증류수 5 L에 첨가하고 100℃에서 7시간 추출한 후 여과시켰다. 이 때, 1차 여과는 2 ㎛ 필터로, 2차 여과는 0.45 ㎛ 필터로, 3차 여과는 0.2 ㎛ 필터로 수행하였다. 1 kg of dried antler was crushed using a crusher. 1 kg of crushed antler powder was added to 5 L of distilled water, and extracted at 100 ° C. for 7 hours and filtered. At this time, the primary filtration was performed by a 2 μm filter, the secondary filtration by a 0.45 μm filter, and the tertiary filtration by a 0.2 μm filter.
<실험예 1> Experimental Example 1 생리활성물질의 분석Analysis of Bioactive Substances
1) 질소총량 및 아미노산1) Total nitrogen and amino acid
상기 실시예 1 및 비교예 1, 2에서 최종적으로 얻어진 녹용 추출물의 질소 총량을 분석하였다. 결과는 다음 표 1과 같다. The total amount of nitrogen of the antler extract finally obtained in Example 1 and Comparative Examples 1 and 2 was analyzed. The results are shown in Table 1 below.
표 1
항목 아미노산 분석결과
실시예1 비교예1 비교예2
질소총량 8.78 0.78 0.15
Table 1
Item Amino acid analysis
Example 1 Comparative Example 1 Comparative Example 2
Nitrogen 8.78 0.78 0.15
(질소총량 및 아미노산 분석)                (Total nitrogen and amino acid analysis)
상기 표 1에 나타난 바와 같이, 본 발명에 따른 발효녹용 분자캡슐은 종래의 방법으로 얻어진 녹용 추출물(비교예 1 및 2)에 비해 질소총량과 아미노산 함량이 수배 내지 수십 배 증가된 것을 알 수 있다.As shown in Table 1, the fermented antler molecular capsule according to the present invention can be seen that the total nitrogen and amino acid content is increased several times to several tens of times compared to the antler extract (Comparative Examples 1 and 2) obtained by the conventional method.
2) 생리활성물질 분석2) Bioactive substance analysis
① 단백질 정량① Protein quantification
100 ug/ml의 표준 소혈청 알부민 (BSA) 용액에서 20, 40, 60, 80, 100 ul를 취하여 적정량의 증류수 (각각 780, 760, 740, 720, 700 ul) 및 200 ul의 단백질 검정 염료를 혼합하여 1 ml로 만들고, 이를 595 nm에서 UV 분광 광도계를 이용해서 흡광도를 측정하였다. 시료를 증류수에 녹여 단백질 검정 염료를 섞어 1 ml로 만들고, 이를 595 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하고 BSA에 대한 함유량으로 계산하여 함량을 결정하였다. Take 20, 40, 60, 80, and 100 ul of 100 ug / ml standard bovine serum albumin (BSA) solution to obtain an appropriate amount of distilled water (780, 760, 740, 720, 700 ul) and 200 ul of protein assay dye. The mixture was made to 1 ml, and the absorbance was measured using a UV spectrophotometer at 595 nm. The sample was dissolved in distilled water, mixed with protein assay dye to 1 ml, and the content was determined by measuring absorbance at 595 nm using a UV spectrophotometer and calculating the content for BSA.
② 우론산 정량 (Carbazole assay)② Determination of uronic acid (Carbazole assay)
D-글루쿠론산 락톤을 표준물로 사용하여 카르바졸(carbazole) 반응에 의해 530 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하였다.Absorbance was measured using a UV spectrophotometer at 530 nm by carbazole reaction using D-glucuronic acid lactone as a standard.
시료를 증류수에 녹여 카르바졸 반응에 의해 530 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하고 D-글루쿠론산 락톤에 대한 함유량으로 계산하여 함량을 결정하였다.The sample was dissolved in distilled water and the absorbance was measured at 530 nm by carbazole reaction using a UV spectrophotometer, and the content was determined by calculating the content for D-glucuronic acid lactone.
③ 글리코사미노글리칸 정량 (Carbazole assay)③ Determination of glycosaminoglycans (Carbazole assay)
NZP 콘드로이틴 설페이트를 표준물로 사용하여 카르바졸 반응에 의해 530 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하였다.Absorbance was measured using a UV spectrophotometer at 530 nm by carbazole reaction using NZP chondroitin sulfate as a standard.
시료를 증류수에 녹여 카르바졸 반응에 의해 530 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하고 NZP 콘드로이틴 설페이트에 대한 함유량으로 계산하여 함량을 결정하였다.The sample was dissolved in distilled water and the absorbance was measured using a UV spectrophotometer at 530 nm by carbazole reaction, and the content was determined by calculating the content for NZP chondroitin sulfate.
④ 시알산 정량 (Warren assay)④ Sialic acid quantification (Warren assay)
N-아세틸뉴라민산을 표준물로 사용하여 Warren 방법에 의해서 549 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하였다.Absorbance was measured using a UV spectrophotometer at 549 nm by Warren method using N-acetylneuraminic acid as a standard.
시료를 80 ℃에서 1 시간 동안 0.1 N 황산으로 가수분해한 후에 Warren 방법에 의해서 549 nm에서 UV 분광 광도계를 이용하여 흡광도를 측정하고 N-아세틸뉴라민산에 대한 함유량으로 계산하여 함량을 결정하였다.After the sample was hydrolyzed with 0.1 N sulfuric acid at 80 ° C. for 1 hour, the absorbance was measured at 549 nm using a Warren method using a UV spectrophotometer, and the content was determined by calculating the content for N-acetylneuraminic acid.
⑤ 효소에 의한 콘드로이틴 설페이트의 분해⑤ Degradation of Chondroitin Sulfate by Enzyme
프로테우스 불가리스(Proteus vulgaris)에서 유래한 콘드로이티나제 (Chondroitinase) ABC (C2905, Sigma)를 50 mM Tris-HCl 60 mM 소듐 아세테이트 완충제 (pH 8.0)에 녹여서 1U/ml 되게 하였다. NZP CS를 표준물로 사용하여 표준물과 샘플을 10 mg/ml로 녹여서 완충제와 섞어 효소를 30 mU 넣고, 이를 37 ℃에서 12시간 반응하여 콘드로이틴 설페이트의 함유량을 분석하였다.Chondroitinase ABC (C2905, Sigma) from Proteus vulgaris was dissolved in 50 mM Tris-HCl 60 mM sodium acetate buffer (pH 8.0) to 1 U / ml. Using NZP CS as a standard, the standard and the sample were dissolved at 10 mg / ml, mixed with a buffer, 30 mU of enzyme, and reacted at 37 ° C. for 12 hours to analyze the content of chondroitin sulfate.
⑥EGF의 분석⑥ Analysis of EGF
EGF의 분석을 위해 녹용열수추출물, 가수분해물, 발효추출물에 대해 사람의 EGF 특이적인 ELISA를 수행하였다. 표준 곡선 작성을 위해 250pg/ml ~ 0pg/ml 농도의 구간에서 선형회귀분석을 통하여 선형성을 갖는 농도구간을 선정하고 이 구간에서 각 3종의 시료를 시험에 적용하여 흡광도 분석을 수행하였다. 이로부터 표준 EGF의 농도에 따른 시료의 EGF를 분석하였다.For the analysis of EGF, human EGF-specific ELISA was performed on antler hydrothermal extract, hydrolyzate and fermented extract. In order to prepare a standard curve, the concentration range having linearity was selected through linear regression analysis in the range of 250 pg / ml ~ 0 pg / ml, and absorbance analysis was performed by applying three samples to the test in this section. From this, the EGF of the sample was analyzed according to the concentration of the standard EGF.
⑦강글리오사이드의 정량(zig-zag TLC scanner)⑦ Quantification of gangliosides (zig-zag TLC scanner)
강글리오사이드 표준물(Sigma Co.)을 메탄올에 용해시킨 후 TLC법에 의해 전개시킨 후 zig-zag TLC 스캐너로 파장 550nm에서 스캐닝하여 얻은 Rf 0.39와 0.42의 피크 적분치로부터 검량선을 작성하였다.A calibration curve was prepared from peak integration values of Rf 0.39 and 0.42 obtained by dissolving the ganglioside standard (Sigma Co.) in methanol, developing by TLC method, and scanning at a wavelength of 550 nm with a zig-zag TLC scanner.
이 후 각 시료를 표준품 방법과 같이 동일한 방법으로 수행하여 Rf 0.39와 0.42의 피크 적분치로부터 각 시료들의 강글리오사이드류를 정량하였다.Thereafter, each sample was performed in the same manner as in the standard method to quantify the gangliosides of each sample from the peak integration values of Rf 0.39 and 0.42.
상기의 분석법을 이용하여 열수추출, 가수분해, 발효추출분자캡슐에 대한 생리활성물질의 분석결과를 하기 표 2에 나타내었다.The analysis results of the biologically active substances for hydrothermal extraction, hydrolysis, fermentation and extraction molecular capsules using the above analysis method are shown in Table 2 below.
표 2
성분시료 단백질 시알산 GAG 우론산 콘드로이틴설폐이트 EGF(μg/g) 강글리오사이드
열수수출 52.2 0.023 5.83 0.245 25.6 0.00024 5.83
가수분해 68.4 0.015 2.67 0.171 35.3 - 2.67
발효녹용분자캡슐 75.8 0.032 9.35 0.374 78.6 0.00057 9.35
TABLE 2
Ingredient protein Sialic acid GAG Uronic acid Chondroitin Sulfate EGF (μg / g) Ganglioside
Hot water export 52.2 0.023 5.83 0.245 25.6 0.00024 5.83
Hydrolysis 68.4 0.015 2.67 0.171 35.3 - 2.67
Fermented Antler Molecular Capsule 75.8 0.032 9.35 0.374 78.6 0.00057 9.35
(녹용추출물별 생리활성물질 분석) 단위(mg/g)               (Analysis of bioactive substances by antler extract) Unit (mg / g)
상기 표 2에서와 같이 열수추출, 가수분해된 녹용추출물보다 발효에 의해 추출되고 이를 분자캡슐화한 것이 생리활성물질이 다량 함유되어 있음을 알 수 있다. As shown in Table 2, the hydrothermal extract, hydrolyzed antler extract, and extracted by fermentation and molecular encapsulation can be seen that the bioactive substance contains a large amount.
<실시예 3><Example 3> 강글리오사이드의Ganglioside 안정성 시험Stability test
녹용의 대표적인 생리활성물질인 강글리오사이드의 열, 산에 대한 안정성 시험을 하였다. 그리나 상기 표 2에서 가수분해에 의한 강글리오사이드의 함량이 제일 낮아 이번 비교실험에서는 제외하였고, 실시예 1, 비교예 2 및 실시예 1에서 분자캡슐하지 않은 발효추출방법으로 제조된 녹용을 80℃에서 4주 동안 보관하는 온도 가혹조건과 pH 1 에서 4주 동안 보관하는 산 가혹조건에서 강글리오사이드의 변화량을 측정하였다. 그 결과는 도 2와 도 3에 표시하였다. Stability test of heat and acid of ganglioside, a representative bioactive substance of deer antler, was performed. However, in Table 2, the content of ganglioside by hydrolysis was the lowest, and thus was excluded from the comparative experiments. Example 1, Comparative Example 2 and Example 1 were prepared by the fermentation extraction method without the molecular capsules 4 at 80 ℃ Changes in gangliosides were measured under severe conditions of temperature storage for one week and acidic conditions for four weeks at pH 1. The results are shown in FIGS. 2 and 3.
도 2의 결과로부터 고온으로 열수추출된 녹용은 시간이 경과됨에 따라 강글리오사이드의 함량이 크게 감소되며 상대적으로 추출온도가 낮은 발효의 경우는 열수추출보다 강글리오사이드 함량이 높음을 알 수 있지만 이 또한 감소됨을 알 수 있었다. 그러나 분자캡슐화된 녹용의 강글리오사이드 함량은 시간이 지나도 초기상태와 같이 그 함량이 크게 변하지 않음을 알 수 있었다.From the results of FIG. 2, the antler extracted from hot water at a high temperature shows a significant decrease in the content of gangliosides over time, and in the case of fermentation with a relatively low extraction temperature, the ganglioside content is higher than that of hot water extraction, but this also decreases. Could. However, the ganglioside content of the molecular encapsulated deer antler did not change much as the initial state over time.
도 3의 결과로부터 고온으로 열수추출된 녹용은 강산 조건에서 시간이 경과됨에 따라 강글리오사이드의 함량이 크게 감소되며 상대적으로 추출온도가 낮은 발효의 경우는 열수추출보다 강글리오사이드 함량이 높음을 알 수 있지만 이 또한 감소됨을 알 수 있었다. 그러나 분자캡슐화된 녹용의 강글리오사이드의 함량은 시간이 지나도 초기상태와 같이 그 함량이 크게 변하지 않음을 알 수 있었다.From the results of FIG. 3, the antler extracted from hot water at high temperature shows a significant decrease in the content of gangliosides over time in strong acid conditions, and in the case of fermentation with a relatively low extraction temperature, the ganglioside content is higher than that of hot water extraction. It can be seen that the decrease. However, the content of the gangliosides of the antler molecule encapsulated in the molecular weight did not change much like the initial state over time.
<실시예 4> 발효녹용 분자캡슐의 항염 활성 시험Example 4 Anti-inflammatory Activity Test of Fermented Rust Molecular Capsules
1)시료 : 발효녹용 분자캡슐1) Sample: Molecular Capsule for Fermentation Rust
2)동물2) animals
실험동물은 대한실험동물센터에서 구입한 Balb/c계 생쥐를 1주일 동안 실험실 환경에 적응시킨 후 실험에 사용하였다. 동물 사육실의 조건은 통상적인 시스템으로 22±2℃, 하루 중 12시간은 200-300 Lux로 조명하고, 12시간은 모든 빛을 차단하였다. 동물에 고형사료 (조단백질 22.1% 이상, 조지방 8.0% 이하, 조섬유 5.0% 이하, 조회분 8.0% 이하, 칼슘 0.6% 이상, 인 0.4% 이상, 삼양사, 항생제 무첨가)와 물을 충분히 공급하였다.Experimental animals were used in the experiment after adapting the Balb / c mice purchased from the Korea Experimental Animal Center in the laboratory environment for one week. The conditions of the animal kennel were a conventional system, 22 ± 2 ° C., 12 hours of the day were illuminated at 200-300 Lux, and 12 hours were all blocked. Animals were supplied with solid feed (more than 22.1% crude protein, less than 8.0% crude fat, less than 5.0% crude fiber, less than 8.0% crude ash, more than 0.6% calcium, more than 0.4% phosphorus, Samyangsa, no antibiotics) and water.
3)시약 및 기기3) Reagents and Devices
본 실험에 사용된 시약중 지질다당류 (LPS)는 Sigma (Sigma Co., USA) 제품을, 생리식염수는 중외제약 제품을, IL-1β, IL-6, TNF-α, MCP-1 ELISA kit는 Millipore사 (USA) 제품을 사용하였으며, 기타 시약은 특급 시약을 사용하였다. 본 실험에 사용된 기기는 원심분리. (Beckman Co., USA), 롤러 혼합. (Gowon scientific technology Co., Korea), 보텍스 혼합.(vortex mixer) (Vision scientific Co., Korea), Luminex (Millipore, Co., USA) 등을 사용하였다.Among the reagents used in this experiment, lipopolysaccharide (LPS) is manufactured by Sigma (Sigma Co., USA), physiological saline is a foreign pharmaceutical product, and IL-1β, IL-6, TNF-α, and MCP-1 ELISA kit Millipore Inc. (USA) was used, and other reagents were used as express reagents. The instrument used in this experiment was centrifuged. (Beckman Co., USA), roller mixing. (Gowon scientific technology Co., Korea), vortex mixer (Vision scientific Co., Korea), Luminex (Millipore, Co., USA) and the like were used.
4)실험방법(in vivo 실험)4) Experimental method ( in vivo experiment)
지질다당류 (LPS)로 유도된 염증 생쥐 모델 및 사이토카인 측정 정상군은 마리당 염수 200 ul를 경구투여하고, 발효녹용 분자캡슐 투여군은 생쥐 마리당 200 mg, 100 mg/kg을 경구용 바늘(oral zonde)을 이용하여 하루에 1회씩 7일간 경구 투여하였다. 7일 후 지질다당류 (LPS) 1 ㎎/㎏을 복강에 주사한 후 60분 후에 에틸 에테르로 마취하고 심장 천자법으로 채혈하였다. 채혈 후 혈청을 분리하여 IL-1β, IL-6, TNF-α, MCP-1 생성량을 ELISA법으로 측정하였다. 각 웰에 생쥐의 혈청 100 ㎕ (1/100 희석)씩 분주한 후 항체-바이오틴 콘주게이트된 100 ㎕를 처리하고 2시간 실온에서 방치한 후 2회 세척 완충 용액으로 세척한 다음 항체 아비딘-HRP 콘주게이트된 100 ㎕를 처리하고 2시간 실온에서 방치한 후 다시 세척하였다. 여기에 TMB 기질을 100 ㎕씩 분주하고 암소에서 30분간 방치한 후 100 ㎕의 스톱(stop) 용액을 처리한 후 ELISA 판독기로 450 nm에서 흡광도를 측정하였다.Inflammatory mouse model and cytokine measurement induced by lipopolysaccharide (LPS) The normal group orally administered 200 ul of saline sugar, and the fermented antler molecular capsule group received 200 mg, 100 mg / kg oral needle (oral zonde) It was administered orally once a day for 7 days using. After 7 days, 1 mg / kg of lipopolysaccharide (LPS) was injected into the abdominal cavity, followed by anesthesia with ethyl ether 60 minutes, and blood was collected by cardiac puncture. Serum was isolated after blood collection and IL-1β, IL-6, TNF-α, and MCP-1 production were measured by ELISA. Dispense 100 μl (1/100 dilution) of mouse serum into each well, treat 100 μl of antibody-biotin conjugated, leave at room temperature for 2 hours, wash with twice wash buffer solution, and then antibody avidin-HRP conjugate Treated 100 μl gated and left for 2 hours at room temperature and washed again. 100 μl of TMB substrate was added thereto, and left in the dark for 30 minutes. After treatment with 100 μl of stop solution, the absorbance was measured at 450 nm with an ELISA reader.
5) 통계처리5) Statistical Processing
다양한 실험으로부터 얻은 결과는 평균 ± 표준편차로 기록하였고, 유의성 검증은 스튜던트(Student's) t-test 분석 방법을 이용하여 결정하였다.Results from various experiments were recorded as mean ± standard deviation, and significance test was determined using Student's t-test analysis method.
6) 결과6) Results
6-1) IL-66-1) IL-6
BALB/c 생쥐에 7일간 발효녹용 분자캡슐을 투여한 후 LPS로 유도된 급성 염증모델에서 혈청을 분리하여 IL-6를 측정한 결과 정상군은 123.2±35.7(pg/ml), 대조군이 544.6±37.1(pg/ml), 발효녹용 분자캡슐 100 투여군은 301.6±56.7(pg/ml), 발효녹용 분자캡슐 200 투여군은 212.2±9.8(pg/ml)로 나타나, 대조군은 정상군에 비해 유의성 있게 증가하였고 발효녹용 분자캡슐 투여군에서는 대조군에 비하여 농도 의존적으로 유의성 있게 (**p<0.01, ***p<0.001) 감소하였다. 그 결과는 도 4에 나타내었다. IL-6 was measured in the LPS-induced acute inflammation model after administration of BALB / c mice for 7 days after fermentation antler capsules. As a result, 123.2 ± 35.7 (pg / ml) in the normal group and 544.6 ± 37.1 (pg / ml), fermented antler molecular capsule 100 administration group showed 301.6 ± 56.7 (pg / ml), fermented antler molecular capsule 200 administration group showed 212.2 ± 9.8 (pg / ml), significantly increased compared to normal group. In the fermented antler molecular capsule group, the concentration-dependency was significantly higher than that of the control group. (** p <0.01, *** p <0.001) decreased. The results are shown in FIG.
6-2) MCP-16-2) MCP-1
BALB/c 생쥐에 7일간 발효녹용 분자캡슐을 투여한 후 LPS로 유도된 급성 염증모델에서 혈청을 분리하여 MCP-1를 측정한 결과 정상군은 124.1± 25.2(pg/ml), 대조군이 418.2±88.1(pg/ml), 발효녹용 분자캡슐 100 투여군은 214.8±31.1(pg/ml), 발효녹용 분자캡슐 200 투여군은 198.2±22.6 (pg/ml)로 나타나 대조군은 정상군에 비해 유의성 있게 증가하였고, 발효녹용 분자캡슐 투여군에서는 대조군에 비하여 농도 의존적으로 유의성 있게 (*p<0.05, **p<0.01) 감소하였다. 그 결과는 도 5 에 나타내었다. After 7 days of fermented antler molecular capsule in BALB / c mice, serum was isolated from LPS-induced acute inflammatory model, and MCP-1 was measured in normal group and 124.1 ± 25.2 (pg / ml) in control group and 418.2 ± in control group. 88.1 (pg / ml), 214.8 ± 31.1 (pg / ml) of fermented antler molecular capsule 100 and 198.2 ± 22.6 (pg / ml) of fermented antler molecular capsule 200 were significantly increased compared to normal group. In the fermented deer antler capsule group, the concentration-dependently decreased (* p <0.05, ** p <0.01) compared with the control group. The results are shown in FIG.
6-3) IL-1b6-3) IL-1b
BALB/c 생쥐에 7 일간 발효녹용 분자캡슐을 투여한 후 LPS로 유도된 급성 염증모델에서 혈청을 분리하여 IL-1b를 측정한 결과 정상군은 25.6±5.6(pg/ml), 대조군이 116.8±8.6(pg/ml), 발효녹용 분자캡슐 100 투여군은 61.2±11.1(pg/ml), 발효녹용 분자캡슐 200 투여군은 51.6±9.2(pg/ml)로 나타나 대조군은 정상군에 비해 유의성 있게 증가하였고 발효녹용 분자캡슐 투여군에서는 대조군에 비하여 농도 의존적으로 유의성 있게 (**p<0.01, ***p<0.001) 감소하였다. 그 결과는 도 6 에 나타내었다. IL-1b was measured in LPS-induced acute inflammation model after administration of fermented antler molecular capsules to BALB / c mice for 7 days, and 25.6 ± 5.6 (pg / ml) in control group and 116.8 ± in control group 8.6 (pg / ml), 61.2 ± 11.1 (pg / ml) of fermented antler molecular capsule 100 and 51.6 ± 9.2 (pg / ml) of fermented antler molecular capsule 200 were significantly increased compared to normal group. In the fermented deer antler capsule group, the concentration-dependence was significantly higher than that of the control group. (** p <0.01, *** p <0.001) decreased. The results are shown in FIG.
6-4) TNF-a6-4) TNF-a
BALB/c 생쥐에 7일간 발효녹용추출분말을 투여한 후 LPS로 유도된 급성 염증모델에서 혈청을 분리하여 TNF-a를 측정한 결과 정상군은 131.4±36.0(pg/ml), 대조군이 397.0±91.1(pg/ml), 발효녹용 분자캡슐 100 투여군은 311.4±54.5(pg/ml), 발효녹용 분자캡슐 200 투여군은 143.2±32.5 (pg/ml)로 나타나 대조군은 정상군에 비해 유의성 있게 증가하였고 발효녹용 분자캡슐 투여군에서는 대조군에 비하여 농도 의존적으로 유의성 있게 ***p<0.001) 감소하였다. 그 결과는 도 7 에 나타내었다.After administration of fermented antler extract powder to BALB / c mice for 7 days, serum was isolated from LPS-induced acute inflammation model, and TNF-a was measured in normal group and 137.0 ± 36.0 (pg / ml). 91.1 (pg / ml), the fermented antler molecular capsule 100 group was 311.4 ± 54.5 (pg / ml), and the fermented antler molecular capsule 200 group was 143.2 ± 32.5 (pg / ml). In the fermented deer antler capsule group, the concentration-dependence was significantly higher than that of the control group. *** p <0.001) decreased. The results are shown in FIG.
본 발명은 가장 자연친화적인 발효방법으로 녹용을 추출하였고 녹용의 생리활성 물질의 파괴를 줄이고 유효성분의 함량을 증가시키며 생산성 또한 높일 수 있어, 다양한 분야에서 사용되는 녹용 추출물의 제조 방법으로 유용하게 이용될 수 있다. 특히 본 발명은 항염 활성을 체계적으로 밝힘으로써 식품, 화장품 등에 널리 사용하기에 적합하다. The present invention can extract the antler by the most natural fermentation method and reduce the destruction of physiologically active substances of the antler, increase the content of the active ingredient and also increase the productivity, it is usefully used as a manufacturing method of the antler extract used in various fields Can be. In particular, the present invention is suitable for wide use in food, cosmetics, etc. by systematically revealing the anti-inflammatory activity.

Claims (17)

  1. 세절된 녹용에 정제수를 첨가하고 열수 추출 및 여과하는 단계(단계 1);Adding purified water to the sliced antler, followed by hot water extraction and filtration (step 1);
    단계 1에서 얻어진 액상분에, 세절된 녹용 100 중량부 당 10 내지 30 중량부의 바실러스 균주 배양액을 첨가하고 30 내지 40℃에서 2 내지 3일 동안 발효시켜 제 1 발효 추출액을 수득하는 단계(단계 2); 및To the liquid powder obtained in step 1, adding 10 to 30 parts by weight of a Bacillus strain culture solution per 100 parts by weight of chopped antler and fermenting at 30 to 40 ° C. for 2 to 3 days to obtain a first fermentation extract (step 2) ; And
    단계 1에서 얻어진 녹용박에 정제수를 첨가한 후에, 세절된 녹용 100 중량부 당 10 내지 30 중량부의 아스퍼질러스 균주 배양액을 첨가하고 50 내지 70℃에서 2 내지 3일 동안 발효시켜 제 2 발효 추출액을 수득하는 단계(단계 3)를 포함하는, 녹용으로부터 녹용 발효 추출물을 제조하는 방법.After the purified water was added to the antler obtained in step 1, 10 to 30 parts by weight of Aspergillus strain culture solution was added per 100 parts by weight of the sliced antler and fermented at 50 to 70 ° C. for 2 to 3 days to obtain the second fermentation extract. Method of producing a antler fermentation extract from antler, comprising the step of obtaining (step 3).
  2. 제 1항에 있어서, 상기 제 1 발효 추출액, 제 2 발효 추출액, 및 제 1 발효 추출액 및 제 2 발효 추출액의 혼합물로 이루어진 군으로부터 선택된 하나 이상에 포접 복합체를 첨가한 후에, 교반하여 분자캡슐화시키는 단계를 추가로 포함하는 방법.The method of claim 1, wherein after the inclusion complex is added to at least one selected from the group consisting of the first fermentation extract, the second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract, stirring and molecular encapsulation How to further include.
  3. 제 1항 또는 제 2항에 있어서, 상기 단계 1에서, 세절된 녹용 100 중량부 당 200 내지 500 중량부의 정제수가 첨가되며, 열수 추출이 80 내지 100℃에서 수행됨을 특징으로 하는 방법.The method according to claim 1 or 2, wherein in step 1, 200 to 500 parts by weight of purified water is added per 100 parts by weight of chopped antler, and hot water extraction is performed at 80 to 100 ° C.
  4. 제 1항에 있어서, 바실러스 균주 배양액이 바실러스 카제이(Bacillus casei)를 함유함을 특징으로 하는 방법.2. The method of claim 1, wherein the Bacillus strain culture contains Bacillus casei .
  5. 제 1항에 있어서, 아스퍼질러스 균주 배양액이 아스퍼질러스 카제이(aspergillus casei)를 함유함을 특징으로 하는 방법.The method of claim 1, wherein the Aspergillus strain culture medium is Aspergillus casei (aspergillus                                  casei).
  6. 제 2항에 있어서, 제 1 발효 추출액, 제 2 발효 추출액, 및 제 1 발효 추출액 및 제 2 발효 추출액의 혼합물로 이루어진 군으로부터 선택된 하나 이상에 이러한 혼합물 100 중량부 당 20 내지 30 중량부의 포접 복합체를 첨가한 후에, 60 내지 70℃에서 교반하고, 이를 상온까지 서서히 냉각시켜 분자캡슐화 시킴을 특징으로 하는 방법.The inclusion complex of claim 2, wherein at least one selected from the group consisting of a first fermentation extract, a second fermentation extract, and a mixture of the first fermentation extract and the second fermentation extract is mixed with 20 to 30 parts by weight of the inclusion complex After addition, the method is characterized in that the mixture is stirred at 60 to 70 DEG C, and gradually cooled to room temperature to encapsulate the molecule.
  7. 제 8항에 있어서, 포접 복합체가 사이클로덱스트린, 크라운 에테르, 폴리옥시알킬렌, 프로포린, 폴리실록산, 포파젠, 및 제올라이트로부터 선택된 것을 특징으로 하는 방법.9. The method of claim 8, wherein the clathrate complex is selected from cyclodextrins, crown ethers, polyoxyalkylenes, proporines, polysiloxanes, popazenes, and zeolites.
  8. 세절된 녹용을 열수 추출하여 얻어진 액상분에 바실러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과 포접 복합체로부터 형성된 복합체.A complex formed from a fermentation extract and an inclusion complex obtained by adding and fermenting a Bacillus strain culture medium to a liquid powder obtained by hot water extraction of finely divided antler.
  9. 세절된 녹용을 열수 추출하여 얻어진 고형의 녹용분에 아스퍼질러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과 포접 복합체로부터 형성된 복합체.A complex formed from a fermentation extract and an inclusion complex obtained by adding and fermenting an Aspergillus strain culture solution to a solid antler obtained by hot water extraction of finely divided antler.
  10. 세절된 녹용을 열수 추출하여 얻어진 액상분에 바실러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액과, 세절된 녹용을 열수 추출하여 얻어진 고형의 녹용분에 아스퍼질러스 균주 배양액을 첨가 및 발효시켜 얻어진 발효 추출액의 혼합물, 및 포접 복합체로부터 형성된 복합체.Fermentation extract obtained by adding and fermenting Bacillus strain culture liquid to liquid powder obtained by hot water extraction of finely divided antler, and fermentation extract obtained by adding and fermenting Aspergillus strain culture solution to solid antler obtained by hot water extraction of finely divided antler. Mixtures, and complexes formed from inclusion complexes.
  11. 제 8항 내지 제 10항 중 어느 한 항에 있어서, 포접 복합체가 사이클로덱스트린, 크라운 에테르, 폴리옥시알킬렌, 프로포린, 폴리실록산, 포파젠, 및 제올라이트로부터 선택된 것을 특징으로 하는 복합체.The complex according to any one of claims 8 to 10, wherein the clathrate complex is selected from cyclodextrin, crown ether, polyoxyalkylene, proporin, polysiloxane, popazene, and zeolite.
  12. 제 2항에 따라 얻어진 생성물, 또는 제 10항 내지 제 12항 중 어느 한 항의 복합체를 고압 멸균시켜 얻어진 액상형의 녹용 발효 추출물.A liquid type antler fermented extract obtained by autoclaving the product obtained according to claim 2 or the complex of any one of claims 10 to 12.
  13. 제 2항에 따라 얻어진 생성물, 또는 제 10항 내지 제 12항 중 어느 한 항의 복합체를 고압 멸균시키고 이를 동결건조시켜 얻어진 분말형의 녹용 발효 추출물.A powdered deer antler fermentation extract obtained by autoclaving and lyophilizing the product obtained according to claim 2 or the complex of any one of claims 10 to 12.
  14. 제 8항 또는 제 10항에 있어서,The method of claim 8 or 10,
    상기 바실러스 균주 배양액에 함유되는 발효균주는, 바실러스 카제이(Bacillus casei , 수탁번호 KACC 91310P ), 바실러스 서브틸리스(B. subtilis), 바실러스 나토(B. natto), 바실러스 브레비스(B. brevis) 중에서 선택된 어느 하나인 것을 특징으로 하는 복합체.Fermented strains contained in the Bacillus strain culture solution, Bacillus casei (Bacillus                                  casei                 , Accession number                 KACC                 91310P), Bacillus subtilis (B.                 subtilis), Bacillus NATO (B.                 natto), Bacillus brevis (B.                 brevisComplex) characterized in that any one selected from.
  15. 제 9항 또는 제 10항에 있어서,The method according to claim 9 or 10,
    상기 아스퍼질러스 균주 배양액에 함유되는 발효균주는 아스퍼질러스 카제이(aspergillus casei), 아스퍼질러스 오리재(A. oryzae), 아스퍼질러스 나이거(A. niger), 아스퍼질러스 글라우쿠스(A. glaucus), 그중에서도 아스퍼질러스 나이거(aspergillus niger) 중에서 선택된 어느 하나인 것을 특징으로 하는 복합체.Fermented strains contained in the Aspergillus strain culture solution Aspergillus Kaze (aspergillus                                  casei), Aspergillus duckA.                 oryzae), Aspergillus Niger (A. niger), Aspergillus Glaucus (A.                 glaucus), Among them Aspergillus Niger (aspergillus                                  nigerComplex) characterized in that any one selected from.
  16. 제 1항 또는 제 2항에서 제조된 녹용 발효 추출물,Deer antler fermented extract prepared in claim 1,
    제 8항 내지 제 10항 중 어느 한 항의 복합체,The complex of any one of claims 8 to 10,
    제 2항에 따라 얻어진 생성물, 또는 제 10항 내지 제 12항 중 어느 한 항의 복합체를 고압 멸균시켜 얻어진 액상형의 녹용 발효 추출물, 및Liquid antler fermentation extract obtained by autoclaving the product obtained according to claim 2 or the complex of any one of claims 10 to 12, and
    제 2항에 따라 얻어진 생성물, 또는 제 10항 내지 제 12항 중 어느 한 항의 복합체를 고압 멸균시키고 이를 동결건조시켜 얻어진 분말형의 녹용 발효 추출물로부터 선택된 것 중 하나 이상을 유효성분으로 함유한 기능성 건강식품.Functional health containing at least one selected from the product obtained according to claim 2, or a powdered antler fermentation extract obtained by autoclaving the complex of any one of claims 10 to 12 and lyophilizing it as a functional ingredient. food.
  17. 제 1항 또는 제 2항에서 제조된 녹용 발효 추출물,Deer antler fermented extract prepared in claim 1,
    제 8항 내지 제 10항 중 어느 한 항의 복합체, 및The complex of any one of claims 8 to 10, and
    제 2항에 따라 얻어진 생성물, 또는 제 10항 내지 제 12항 중 어느 한 항의 복합체를 고압 멸균시키고 이를 동결건조시켜 얻어진 분말형의 녹용 발효 추출물로부터 선택된 것 중 하나 이상을 유효성분으로 함유한 기능성 화장품.Functional cosmetics containing as an active ingredient at least one selected from the products obtained according to claim 2 or the powder of antler fermentation extract obtained by autoclaving the complex of any one of claims 10 to 12 and lyophilizing it. .
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