KR20130119224A - Preparing method for deer bone extract - Google Patents

Abstract

PURPOSE: A production method of a deer bone extract is provided to increase the contents of ganglioside and protein by the enzyme decomposition of a hot water extract deer bones. CONSTITUTION: Blood of deer bones are removed using cold water and warm water. The deer bones are extracted by pressurizing and by using hot water. The hot water extract is filtered, and oil is separated from the filtrate. The separated liquid is high temperature cooked for deodorizing. The separated liquid is enzyme decomposed. Enzymes are deactivated. The enzyme decomposed liquid is concentrated by pressurizing. [Reference numerals] (AA) Removing blood in cold water; (BB) Removing blood in hot water; (CC) Pressurizing and extracting; (DD) Filtering; (EE) Separating oil; (FF) Cooking at high temperature; (GG) Decomposing enzyme; (HH) Deactivating enzyme; (II) Concentrating; (JJ) Drying

Description

Preparation method of green bone extract {PREPARING METHOD FOR DEER BONE EXTRACT}

The present invention is a method for producing green bone extract, hot water extract, hot cooking and enzymatic decomposition to remove the odor unique to the green bone, the method of producing green bone extract to promote growth, joint health, memory improvement, immunity enhancement activity It is about.

Deer bones are called "sacred bones" and are said to be able to treat any kind of disease that is caused by weakness of the whole body, weakness of muscular function due to boiling, ingestion, warmness, taste, and poison.

 Recently, deer's by-products have been found to contain functional ingredients in flesh and bone, and in particular, deer bone contains ganglioside, which is known to be contained only in antler, contains about 40% of antler . Ganglioside was found to contain 5.66 mg / g of ganglioside, 0.77 to 2.87 mg / g of ganglioside in male bark, and 0.92 to 2.31 mg / g of ganglioside in elk deer bone Respectively. Ganglioside is known to be a body-function-maintaining active substance which is known to activate brain cells together with hemoglobin to improve brain function, to improve memory and concentration, and to increase immunity. Recent clinical data have also reported improvements in Parkinson's symptoms and relief of diabetic peripheral neuropathy.

 The green algae extract contains large amounts of protein and phospholipid, which are the main components involved in skeletal formation and metabolism. It also contains chondroitin and collagen, which can help bone and joint health. It also contains minerals such as calcium and phosphorus to enhance nerve strengthening and metabolism.

Studies on green bones include the method of extracting gangliosides using deer meat and bone (concentration method using deer meat and bone and food using the same, Application No. 10-2002-0064443), extraction method to remove odor of green bones containing herbal medicine Method for preparing green bone extract containing extract, and green bone extract prepared by the same, and food and medicine containing the same, Application No. 10-2004-0030312), Extraction method with antler (Health beverage containing antler extract and its Manufacturing method, Application No. 10-2009-0007905), Deer Antler, Venison, Method of extracting a mixture of deer bones (Deer Jungtang Extract manufacturing method, Application No. 10-2009-0126427), etc., but using the green bone alone By optimizing the extraction of both ganglioside and protein to effectively remove the fish odor of golgol, and to enhance the functionality of the extract Phrase is the situation does not already exist.

The present invention is to remove the odor of the golgol by hot water extraction and hot cooking of the golgol, and by increasing the content of ganglioside and protein by enzymatic decomposition of the extract, golgol extract with high growth promoting, joint health, memory improvement, immunity enhancing activity It relates to a manufacturing method of.

The present invention comprises the steps of removing the blood of the golgol with cold water; Removing the blood of the green bone with hot water; Pressurizing the green bone; Filtering the hydrothermal extract; Oil-separating the filtrate: hot deodorizing the oil-separated solution; Enzymatically decomposing the separated liquid; Inactivating the enzyme; It provides a method for producing green bone extract comprising the step of concentrating the enzyme digestion liquid under pressure, drying the concentrated liquid under pressure.

Here, the step of removing the blood with cold water may be performed by mixing green bone and purified water in a weight ratio of 1: 1.3 to 3 and repeating 2-4 times for 2 to 12 hours.

In addition, the step of removing the blood with hot water may be mixed in a weight ratio of 1: 1.3 to 3 of the green bone and purified water and proceed for 20 to 60 minutes under the conditions of 80 ~ 100 ℃.

In addition, the pressure extraction step may be a mixture of green bone and purified water in a weight ratio of 1: 1.5 ~ 2.5 and may proceed for 4-12 hours at 0.5 ~ 1.5 atm at 100 ~ 130 ℃.

In addition, the filtration step may be made of 20 to 60 mesh (mesh).

In addition, the step of separating the oil may be carried out under the conditions of the feed rate 700 ~ 1000 L / hr, 5,000 ~ 8,000 rpm using a three-phase separator.

In addition, the high temperature cooking may be performed for 20 to 60 minutes at a temperature of 200 ~ 250 ℃.

In addition, the enzyme is not particularly limited as long as it is an enzyme that can be suitably used for the proteolysis of the green bone, which acts inside the peptide constituting the green bone as endopeptides, and decomposes the protein, Alcalase, Neutrase, Protamex, Maxazyme NNP, Prowin It may be at least one selected from the group consisting of L20, Promod 278, Protease N, Delvolase, Bioprotease P. At this time, the enzymes may be added 0.1 to 5% with respect to the solid content of the green bone, respectively.

In addition, the enzymatic decomposition may be performed for 1 to 12 hours at 40 ~ 65 ℃ while stirring at 30 ~ 350rpm.

In addition, the step of inactivating the enzyme may be performed for 10 to 50 minutes at 70 ~ 95 ℃.

In addition, the step of concentrating may be carried out under the conditions of 10 ~ 40mmHg at 35 ~ 70 ℃.

Hereinafter, a method for preparing a green tea extract of the present invention will be described in detail.

Step 1: Remove the blood from the alveoli

Remove the blood from the bone as much as possible through the blood of the bone.

First, melt the green bone and purified water at a ratio of 1: 1.3 to 3, and remove the blood of the green bone for 2 to 12 hours. Repeat this process 2-4 times. Secondly, the green bone and purified water which performed the first blood draining are mixed at a weight ratio of 1: 1.3 to 3, and blood is removed by hot water for 20 to 60 minutes under conditions of 80 to 100 ° C.

Second Step: Decompression Hot Water Extraction Step

In the first step, the green bone and purified water, which were extracted with blood, were mixed at a weight ratio of 1: 1.5 to 2.5, and extracted at 100 to 130 ° C. at 0.5 to 1.5 atm for 4-10 hours. Raw materials extracted within these conditions were efficient in terms of stability and yield.

Third Step: Filtration Step

The extract is filtered using a Vibro shifter of 20 to 60 mesh filter network.

Step 4: Oil Separation Step

The filtrate is oil-separated at a feed rate of 700 to 1000 L / hr and 5,000 to 8,000 rpm through a three-phase separator to remove the fat layer.

5th step: high temperature cooking step

Hot cooking the oil separation solution for 20 to 60 minutes at a temperature of 200 ~ 250 ℃ using a high temperature cooker. When cooking the oil separated at high temperature, dimethylamine, which is a rancid component of meat, may be volatilized to remove fishy odor.

6th step: enzyme digestion step

The oil separation from which the fish odor is removed is enzymatically digested at 40-65 ° C. for 1-12 hours while stirring at 30-350 rpm with endopeptides.

Any enzyme that can be used for endopeptides to decompose proteins by acting inside the peptides constituting the golgol is not particularly limited, but Alcalase, Neutrase, Protamex, Maxazyme NNP, Prowin L20, Promod 278, Protease N, Delvolase, Bioprotease P may be one or more selected from the group consisting of. At this time, the enzyme may be added 0.1 to 5% with respect to the solid content of the green bone respectively.

Step 7: enzyme deactivation step

The enzyme digested product to which the endopeptides were added was placed at 70 to 95 ° C. for 10 to 50 minutes to inactivate the endopeptides.

8th step: concentration step

Enzyme digest is concentrated at 35-70 ° C., 10-40 mmHg.

9th Step: Drying Step

The concentrated enzyme decomposition solution may be dried and pulverized. In this case, there is no limitation on the drying method, and general drying methods such as vacuum drying, hot air drying, spray drying and freeze drying are used.

As described above, the present invention removes the characteristic fishy odor by hot water extraction and hot cooking of the green bone, and separates the solid phase and the liquid phase obtained by hydrolysis with an endopeptidase enzyme, and then, the liquid phase is filtered and powdered. Characterized in that the present invention provides a method for producing green bone extract containing growth promoting, joint health, memory improvement, immune enhancing activity factor.

According to the present invention, it is possible to remove the fishy odor peculiar to the golgol, to extract the ganglioside and protein content of the golgol optimally, can be manufactured through a simple process in a short time at a low price, high productivity, growth promotion, Green bone extract, which contains a large amount of joint health, memory, and immune enhancing factors, can be obtained.

1 is a flow chart showing a process of powdering after hot water extraction, hot cooking and enzymatic decomposition of green bone.
Figure 2 relates to the change in flavor components after hot cooking of green bone extract.
Figure 3 relates to the joint health activity of green bone extract.
Figure 4 relates to the memory improving activity of green bone extract.
Figure 5 relates to the anticomplement activity of green bone extract.
Figure 6 relates to the cytokine INF-c enhancement effect of green bone extract.
Figure 7 relates to the cytokine IL-2 enhancement effect of green bone extract.

Hereinafter, the present invention will be described in more detail with reference to Examples and the accompanying drawings. The following examples are merely provided to more easily understand the present invention, but the present invention is not limited to the following examples.

Example 1 Preparation of Green Golgol Extract

Put 500kg of green bone in the extraction tank, add 750 L of purified water and leave for 4 hours, then discard the liquid to remove blood with cold water. Repeat the same process three times. After cold water bleeding, add 750 L of purified water to the green bone and perform hot water bleeding (hot water bleeding) at 95 ° C for 30 minutes. After bleeding hot water, 850 L of purified water was added to the green bone and extracted at 1.5 kg for 10 hours. The extract is filtered using a 60 mesh Vibro shifter. The filtrate is subjected to oil separation under a condition of an input rate of 800 L / hr and 6,500 rpm through a three-phase separator. The separated liquid is deodorized by cooking at a temperature of 240 ° C. for 30 minutes using a high temperature cooker. The temperature of the liquid was raised to 55 ° C, and 0.5% of the endopeptides [Alcalase] enzyme was added to the solid, followed by stirring for 3 hours. After the reaction was completed, the enzyme was inactivated by raising the temperature to 90 ° C. and holding for 15 minutes. The recovered liquid was transferred to a concentrator and concentrated to 50% by weight of solid content at 55 ° C. and 30 mmHg. Powdering was carried out using a vacuum dryer. The main components of green bone extract are shown in Table 1.

Main ingredient of green bone extract content Green bone extract Hot water extract Enzyme digestion protein (%) 85 90 Ganglioside
(mg / kg) 1250 1300

In addition, the deodorizing effect of fish odor through hot cooking is shown in FIG.

As shown in Figure 2 can be reduced by the volatilization of dimethylamine (dimethylamine) which is a rancid component of meat (Fig. 2).

Example 2 Growth Promotion Effect of Green Golgol Extract

MC3T3-E1 cells were treated at 37 ° C., 5% using α-MEM (α-Modified Eagle Medium; sodium bicarbonate, 100 U / mL penicillin, 100 μg / mL streptomycin) medium containing 10% Fetal bovine serum (FBS). Cultured under CO ₂ conditions and medium was changed every 3 days. Treatment of colostrum component 100 ㎍ / mL, green bone extract by concentration was measured by MTT assay to measure the degree of reduction of 3- (3,4-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide reagent. The results are shown in Table 2.

Effect of Green Bone Extract on Proliferation of Osteoblasts (MC3T3-E1 Cell) density
(쨉 g / mL) Green bone extract Hot water extract Enzyme digestion 0 100.0 ± 10.5 100.0 ± 3.8 50 98.6 ± 4.5 112.8 ± 13.4 100 95.4 ± 2.6 129.7 ± 9.6 250 86.0 ± 5.2 137.5 ± 12.2 500 72.2 ± 4.2 137.1 ± 9.4 1000 72.7 ± 6.4 117.4 ± 15.3

The osteoblast proliferation effect of colostrum (colostrums, 100 ㎍ / mL) was 118.9 ± 0.54%. According to Table 2, in the case of the golgol extract, only the hot water extraction group showed cytotoxicity at a concentration of 250 ㎍ / mL or more. On the other hand, the endopeptides treated group did not show cytotoxicity, and it was confirmed that osteoblast proliferation was increased than the normal group.

Example 3 Joint Health Effect of Green Gum Extract

After osteoblast differentiation, colostrum component, hot water extract only green bone extract, and enzymatic digestion were treated at a concentration of 100 ㎍ / mL. After incubation for 4 days, the expression of type II collagen was measured by PCR. The results are shown in Fig. Type II collagen, which accounts for more than 50% of cartilage, is the main protein that makes up bones, ligaments, and tendons. According to Figure 3, both hot water extracted only green bone extract (DBP) and enzymatic digestion (HDBP) of the green bone extract was found to promote the expression of type II collagen compared to the positive control group (colostrum component, PC) when 100 ㎍ / mL treatment Could.

Example 4 Memory Improvement Effect of Green Golgol Extract

Underwater labyrinth test for evaluating the spatial memory of experimental animals was to fill the circular pool (150cm in diameter, 60cm in height) with water 30cm high (23 ± 2 ℃) It was placed at 1 cm and the skim milk was removed so that the pedestal was not visible. On the first day of the experiment, the animals were allowed to swim freely in the tank for 60 seconds without the platform. When the experimental animals reached the platform position, they were allowed to stay on the platform for 10 seconds. If the platform was not found within 120 seconds, the animals were placed on the platform for 10 seconds and remembered. The experiment was repeated every 20 minutes, and the effects of hot water extraction of green bone extract (DBP) and enzymatic digest (HDBP) of green bone extract on the spatial learning of rats that caused memory damage with scopolamine were tested. The results are shown in Fig.

According to Figure 4, the results of cognitive training twice a day for 4 days, tacrine treatment group (2 mg / kg) dementia treatment and hot water extract only green bone extract (DBP, 200 mg / kg) and enzyme decomposition products of the green bone extract ( HDBP, 200 mg / kg) treated group showed an improvement in memory damage by scopolamine from day 2 of cognitive training.

Example 5 Immunity Enhancement Effect of Green Golgol Extract

Anti-complement activity was measured by complement fixation test based on the degree of erythrocyte hemolysis by complement remaining after complement consumption by the sample. 50 μL of normal human serum (NHS) and GVB ++ (gelatin veronal buffered saline, pH 7.4, 2% gelatin, 500 μM Ca ++, 2 mM Mg ++) buffer were mixed in a sample dissolved in distilled water at 37 ° C. The reaction was first performed for 30 minutes, and 350 μL of GVB ++ was added, and then serially diluted 10-160 times. 750 μL of GVB ++ was added thereto, followed by 250 μL of positive sensitized blood cells (IgM-sensitization sheep erythrocyte, EA Cell, 1 × 10 ⁸ cell / mL), followed by secondary reaction at 37 ° C. for 60 minutes, and 2.5 mL of PBS was added thereto. The reaction was stopped. The reaction solution was centrifuged at 2,500 rpm for about 10 minutes, and the supernatant was measured for absorbance at 412 nm to measure the residual hemolytic activity. Anti-complement activity was expressed as inhibition of 50% total complement hemolysis (ITCH ₅₀ ,%) of the control group (50% total complement hemolysis, TCH ₅₀ ) of the control group reacted only with NHS, buffer, and distilled water. The results are shown in Fig. According to FIG. 5, although the activity was slightly lower than that of the polysaccharide-K (PSK) of the cloud control, the positive control group, the green tea extract (DBP) and the enzymatic digest (HDBP) of the green tea extract were excellent anti-complements. It was confirmed that there is activity.

In addition, the psychocaine concentration was measured by ELISA method. Goat anti-mouse cytokine primary antibody was incubated overnight at 4 ° C. using a coating buffer, and then bloking at room temperature for 2 hours with 3% BSA solution. Samples were each dispensed onto plates and incubated at 37 ° C for 2 hours, after which biotinylated anti-cytokine secondary antibodies were added. In addition, an appropriate amount of avidin-conjugated alkaline phosphate was added, incubated at 37 ° C for 2 hours, p-nitrophenyl phosphate was added as a substrate, and the absorbance was measured at 410 nm and 450 nm with a microplate reader. The results are shown in FIGS. 6 and 7. As a result, the cytokines INF-c and IL-2 were treated when the green bone extract (DBP, ○) and the hydrolyzate of the green bone extract (HDBP, ▼) were extracted only at 1,000 ㎍ / mL. Both showed increasing trends (FIGS. 6 and 7).

Claims (13)

Removing the blood of the green bone with cold water;
Removing the blood of the green bone with hot water;
Extracting pressurized hot water from the green bone;
Filtering the hydrothermal extract;
Oil-separating the filtrate:
Hot deodorizing the oil separation solution;
Enzymatically decomposing the separated liquid;
Inactivating the enzyme; And
Method for producing green bone extract comprising the step of concentrating the enzyme decomposition liquid. The method of claim 1,
The step of removing the blood with cold water is mixing the green bone and purified water in a weight ratio of 1: 1.3 to 3 and the method of producing green bone extract is repeated 2-4 times for 2 to 12 hours. The method of claim 1,
The step of removing the blood with hot water is a method for producing green bone extract, which is mixed in a weight ratio of 1: 1.3 to 3 of green bone and purified water and proceeds for 20 to 60 minutes under conditions of 80 to 100 ° C. The method of claim 1,
The pressure extraction step is to mix the green bone and purified water in a weight ratio of 1: 1.5 ~ 2.5 and the production method of the green bone extract proceeds for 4-12 hours at 0.5 ~ 1.5 atm at 100 ~ 130 ℃. The method of claim 1,
The filtration step is a method for producing green bone extract consisting of 20 ~ 60 mesh (mesh) filter. The method of claim 1,
The step of separating the oil is a method for producing green bone extract that is carried out under conditions of a feed rate of 700 ~ 1000 L / hr, 5,000 ~ 8,000 rpm using a three-phase separator. The method of claim 1,
The high temperature cooking is a method for producing green bone extract that proceeds for 20 to 60 minutes at a temperature of 200 ~ 250 ℃. The method of claim 1,
The enzyme is endopeptides, 0.1 to 5 parts by weight based on 100 parts by weight of golgol solids is added to the method of producing golgol extract. 9. The method of claim 8,
The endopeptides are Alcalase, Altrase, Neutrase, Protamex, Maxazyme NNP, Prowin L20, Promod 278, Promod 278, Propro Protease N (Protease N), Delvolase (Delvolase), Bioprotease P (Bioprotease P) A method for producing a green bone extract selected from the group consisting of. The method according to any one of claims 1, 8 and 9,
The step of enzymatic digestion is a method for producing green bone extract, which proceeds for 1 to 12 hours at 40 to 65 ° C. while stirring at 30 to 350 rpm. The method of claim 1,
The step of inactivating the enzyme is a method of producing golgol extract for 10 to 50 minutes at 70 ~ 95 ℃. The method of claim 1,
The step of concentrating is a method of producing green bone extract proceeds under the conditions of 10 ~ 40mmHg at 35 ~ 70 ℃. The method of claim 1,
The method further comprises the step of drying and powdering the filtered enzyme digestion solution of green bone extract.

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