KR101822752B1 - Manufacturing method for enzymatic extracts of deer antlerdeer antler containing highly physiologically active ingredients of deer antler and enzymatic extracts of deer antlerdeer antler using the same - Google Patents

Abstract

The present invention provides a method for manufacturing a hydrolysis extract of deer antler enzyme containing highly physiologically active ingredients of deer antler, which comprises the following steps: a first step (1) for mixing raw deer antler with water in the weight ratio of 1 : 4, boiling the mixture at the temperature of 95C for 7 hours, and concentrating the mixture under reduced pressure to manufacture a deer antler concentrate having 60 to 70 Brix; a second step (2) for mixing a hydrolysis extract to the deer antler concentrate in the step (1) in the weight ratio of 100 : 1 and reacting the mixture for 24 hours with adjusted pH and temperature; and a step (3) for filtering the hydrolyzate of the step (2), removing the enzyme activity, adjusting the pH to 7, and freeze-drying the hydrolyzate. It is an object of the present invention to provide a compounding ratio of a sub ingredient which can maximize the physiological activity of the deer antler extract and facilitate the ingestion thereof.

Description

TECHNICAL FIELD The present invention relates to a method for producing a hydrolysis product of a deer antler containing a physiologically active ingredient of deer antler, and a method for producing the hydrolysis product of a deer antler by hydrolysis of a deer antler, SAME}

The present invention relates to a method for producing a protein and a carbohydrate hydrolase extract which maximizes physiologically active ingredients of deer antler and maximizes the concentration of effective physiologically active ingredients such as uronic acid, sialic acid and collagen And antioxidant properties, and a process for producing the same.

It is said that the antlers are cut and used as a medicine before the horns of the stag are hardened. It has been known from the past that antler has the effect of promoting growth of children with late growth, children with fragile constitution, strengthening the skeleton, improving intelligence development and digestion and absorbing immunity. According to the herbal gangmok, it is known that antler antioxidant has a special effect for anti-aging because it constantly gives vitality to youthful, energizing and skin-protecting effect.

In addition, antler antioxidants help the organs of the body to recover from fatigue, improve sleep and appetite, and have a strong anti-inflammatory action that removes toxins from the body and prevents the warts from getting cold and promotes the production of red blood cells. Anemia patients, before and after childbirth, after a big surgery, it is helpful to take. In addition, antler has a strong effect on muscles, and when there is no strength in the back, knees, and legs, it helps when there are symptoms such as osteoporosis, and it stimulates the secretion of sex hormones. And the effect of stimulating the ovulation of women.

Deer horns vary slightly depending on the type and age of the deer, but they are cut from May to August on average. After turning the horns, the remaining part of the head is hardened and then falls off in the second or third month of the following year. The new horn starts to grow on the spot after it has been withdrawn, and it is folded on the basis of the declining time. In the case of the scarlet, 65 days after declination, 80 days for elk, 70 days for reddish, .

In Korea, many deer are being raised for the production of antler, and the majority of the deer are formed, and the New Zealand mountains belonging to the red deer and the American elk belonging to the big deer are raised, and some are distributed as medicinal products. Elk deer is a large deer deer in red deer. It is introduced in Korea and is a large number of deer among breeders. It is imported from Canada, northern USA, and Russia.

Elk deer is increasingly breeding because of its excellent antler production and self-productivity. The elk deer has antlers that fall from February to March and the new horns begin to sprout. After about 80 to 90 days, the antlers are harvested and harvested. It can be harvested.

However, the consumption of herbal medicine as well as the consumption of antler have also decreased due to the consumption contraction caused by the complicated economic situation such as the recent financial crisis, which leads to a decrease in the number of deer hatching, which threatens the livelihood of the livestock farmers. Despite the fact that antler contains many physiologically active substances and nutrients that contribute to health promotion, it is only consumed with 'hot water extract', 'tablets.' Despite its high nutritional value due to the unique flavor of antler, It is not developed.

In addition, conventional antler antler has been mainly used as a herbal medicine, and has been put into water with other medicines and consumed for a long time by boiling hot water extraction method, and various foods including antler have not been developed. Especially, the method of extracting an effective ingredient by using a high-priced antler extract called hot water extraction has been used, but it has not been used sufficiently although it contains a large amount of excellent functional active ingredients.

In recent years, it has been known that the physiological activity level varies greatly depending on the extraction method, as the method of extracting the physiologically active ingredient from natural products has been diversified due to the development of science. In fact, in the case of bokbunja, the effect of enhancing the anticancer activity and immune activity , And it has been reported that the antioxidant activity is increased by supercritical extraction. Therefore, it is necessary to study the dietary methods of dietary antler extract and to make it easier to ingest.

Korean Patent Registration No. 10-0729513 discloses a method of preparing a decoction concentrate by incorporating predetermined quantities of herbal medicines such as antler, hwanggi, danggu, and taeunggung into predetermined water, Discloses a method for producing a deer antler which can obtain an effective substance. Korean Patent Registration No. 10-0837879 relates to a functional enhanced cosmetic composition comprising the extract of collagen extracted from antler extract and a method for preparing the same, extracting the collagen component from the antler using the crude enzyme extract extracted from the in vitro gold, Discloses a cream and essence composition containing antler-extracted collagen having enhanced antioxidant activity and wrinkle-improving and skin moisturizing-enhancing effect, which have high content of amino acids such as hydrocyclone and hydrocyanic acid, and a method for producing the same. Korean Patent No. 10-1184359 discloses a composition for antioxidant and growth promotion comprising antler antler as an active ingredient fermented with Bacillus subtilis KCTC 11454BP useful for promoting health and promoting growth through antioxidation, Discloses compositions of the present invention. Korean Patent No. 10-0808060 discloses a method for fermenting Bacillus licheniformis (Bacillus licheniformis KCCM10885P), which comprises fermenting Bacillus licheniformis KCCM10885P having a deer antler effect and (a) Bacillus licheniformis KCCM10885P in a culture solution containing deer antler; And (b) concentrating or drying the obtained fermentation broth, thereby producing a fermentation product of green antler using the same, which has excellent antifatigue effect, soybean protecting effect and diarrhea suppressing effect, / RTI > However, the above-mentioned inventions show differences in the method of manufacturing the hydrolysing enzyme-containing extract of the antler of the present invention, which maximizes the effective physiologically active ingredient according to the extraction method according to the antler extract, and its constitution and effect.

The present invention provides a method for extracting antler extract used as a conventional hot water extraction method so as to contain a physiologically active substance using various extraction methods and a hyperglycemic extract extract of the antler extract using the method and maximizing the physiological activity of the antler extract It is an object of the present invention to provide a compounding ratio of a sub-ingredient which can facilitate ingestion.

The present invention relates to a process for producing a deer-friendly concentrate (1) comprising mixing a raw green bean and water at a weight ratio of 1: 4, boiling at 95 ° C for 7 hours and concentrating under reduced pressure to obtain a deer concentration of 60 to 70 ㅀ Brix, (2) mixing the hydrolyzed enzyme with the hydrolysis enzyme at a mass ratio of 100: 1, adjusting the pH and temperature, and allowing to react for 24 hours, filtering the hydrolyzate of step (2) And (3) lyophilization of the dextrin-active component of the deer antler.

The present invention relates to a method for manufacturing a hydrolytic enzyme-containing extract of deer antler according to the present invention, which enables to extract a physiologically active ingredient from an expensive deer antler, It can contribute to industry.

FIG. 1 is an HPLC chromatogram of a deer antler prepared by the antler part. (A: Relative, B: Company, C: Inferior)
Figure 2 shows a schematic diagram of a hydrolytic enzyme treatment process.
FIG. 3 is a photograph showing the hydrolytic enzyme extraction process of the counterpart, middle, and bottom of antler.
4 is a graph showing the content of Uronic acid in each hydrolytic enzyme product of antler.
FIG. 5 is a graph showing the content of sialic acid in each hydrolytic enzyme product of antler.
FIG. 6 is a graph showing the content of glucose aminoglycan in each hydrolytic enzyme product of antler.
7 is a graph showing the content of total carbohydrates in each hydrolytic enzyme product of antler.
FIG. 8 is a graph showing the content of collagen in each hydrolytic enzyme product of antler.
Fig. 9 shows DPPH radical scavenging activity in each antagonist product of antler. (A: graph of DPPH radical scavenging activity B: ESR spectra of flavorzyme treated extracts in antifoaming agent according to each concentration in 60 μM DPPH methanol solution C: ESR spectra)
Fig. 10 shows the hydroxyl radical scavenging activity in each antagonist product of deer antler. (A: Graph of hydroxyl radical scavenging activity, B: ESR spectra of flavorzyme extracts in antler of each concentration in Fenton reaction system)
Fig. 11 shows the alkyl radical scavenging activity in each hydrolytic enzyme product of antler. (A: graph of alkyl radical scavenging activity B: ESR spectra results of each concentration in acerase-treated extracts of antler antagonist observed in 4-POBN-treated AAPH incubation)
12 is a graph showing DPPH radical scavenging activity of each plant extract.

The present invention relates to a process for producing protein and carbohydrate hydrolase extracts maximizing physiologically active ingredients of deer antler, and to an antler dehydrogenase extract using the same. Hereinafter, the present invention will be described in detail with reference to specific examples.

I. Materials and Methods Used in the Invention

1. Manufacture of food ingredients (deer antler and deer antler) using antler

(1) Deer antler powder: The deer antler powder was pulverized and lyophilized.

(2) Deer antler: 4 Kg of water was added to 1 Kg of raw green tea using a household electric kettle (JH-D40, Human Plus), and then extracted at 95 ° C for 7 hours and concentrated under reduced pressure to have a Brix of 60 to 70. Brix.

2. Analysis of general ingredients and effective ingredients of antler processing materials (antler grains and deer antler)

(1) Analysis of general components according to the processed materials: The moisture content was 105 ℃, the crude protein content was measured by Kjeldahl method and the crude fat content by Soxhlet method according to AOAC method (2005).

(2) Analysis of effective ingredients of each antler

A) Uronic acid analysis: A sample of the antler was decalcified at 4 ° C with 0.5M EDTA 2Na (pH 7.4). Crude Papein was mixed with 0.1 M phosphate buffer (pH 6.5) containing 5 mM EDTA and 5 mM Cystein HCl, and Papein was activated for 30 minutes at 65 ° C. Protein was then digested with activated Papein at 65 ° C for 16 hours and then measured at 530 nm by Carbazole reaction (Scott, 1960; Kosakai & Yoshizawa, 1979). At this time, the content was expressed as% by weight with respect to the sample of the antler.

Sialic acid analysis: Nerotonic acid samples are hydrolyzed with 0.1 N sulfuric acid (H2SO4) at 80 ° C for 1 hour and measured at 549 nm by the Warren method (Warren, 1959). At this time, the content was expressed as% by weight with respect to the sample of the antler.

C) Analysis of Glycosaminoglycan: The degradation of papein in uronic acid was measured at 540 nm by the method of dimethymeethylene blue dye binding. At this time, the content was expressed as% with respect to the antler sample.

Total equivalence analysis: Samples A, B and C were diluted 200 and 400 times. glucose standard 100 μg / ml and fructose standard 100 μg / ml were prepared at concentrations of 0, 10, 20, 30, 40 and 50, and 5% sulfuric acid and 95.5% of H ₂ SO ₄ were prepared. standard solution is added to 2 ml of test tube, 1 ml of phenol solution is added, then 5 ml of sulfuric acid is added in a hurry, the tube is vortexed and left for 10 minutes, placed in water for 15 minutes, Absorbance was measured before reacting with oxygen. At this time, the absorbance of the standard should be more than 0.97, and when it is more than 0.97, the sample solution was tested in the same way.

Collagen analysis: Bergman and Loxley (1963) were used to quantify the amount of hydroxyproline. Add 0.1 g of the sample to a screw cap tube, add 10 ml of 6N hydrochloric acid, and hydrolyze at 110 ° C for more than 24 hours. The hydrolyzed solution is concentrated under reduced pressure to remove hydrochloric acid completely and 100 ml of citric acid / After filtering, 100 μl of the sample was diluted in 900 μg of citric acid / acetic acid buffer solution and used as a sample for measuring the amount of hydroxyproline. 300 μl of the sample for measurement and 300 μl of isopropanol are added, and 300 μl of the oxidizing solution is added. After vortexing, 4 ml of Ehrlich's reagent is added, and the sample is heated at 60 ° C. for 25 minutes and then cooled in flowing water for 2-3 minutes. After cooling, colorimetric determination was made at 558 nm within 4 hours, and the amount of collagen was calculated by multiplying collagen coefficient 7.52 by quantified hydroxyproline content.

F) Amino acid analysis: The samples were subjected to acid stabilization pretreatment. That is, about 0.08 g was weighed, and 10 ml of 6N HCl was added thereto, followed by mixing and replacing with nitrogen, followed by sealing and hydrolysis at 110 ° C for 24 hours. The hydrolyzate is filtered (0.45 nm syringe fiter, whatman), filtered under reduced pressure at 55 to 60 ° C, and adjusted to 10 ml (Y) with 0.2 N sodium citrate buffer (pH 2.2). Instrumental analysis was performed using an automatic amino acid analyzer (L-8500A, Hitachi), injected into a capsule with 20 μg (Z), injected into an automatic injector and analyzed by Ninhydrin method at 440 nm and 570 nm.

Amino acid content (%) = M * B / 1,000,000 * 100

M (ng) = A10 (cystine 5) * MW (Mol.Wt)

A (area) = sample area / standard area

B (dilution factor) = (1,000 / X) * (Y / Z)

Z (ml) = (injection amount? (Z) / dilution multiple (B)) / 1,000

3. Analysis of antioxidant activity of antler seeds (antler grains and antler extract concentrates): DPPH, hydroxyl, alkyl and superoxide radical scavenging activity

A) Analysis of DPPH radical scavenging activity: DPPH radical scavenging activity was measured by Nanjo et al. (1996). 60ul of ethanol solution and 60ul of DPPH solution (0.06mM) were mixed with each antler extract and 50ul was transferred into 100ul Teflon capillary tube and used for ESR spectrophotometer analysis. (Measurement condition: central field, 3475 g; modulation frequency, 100 kHz; modulation amplitude, 2 g; microwave power, 5 mW; gain, 6.3 × 10 ⁵ ;

B) Hydroxyl radical scavenging activity analysis: Hydroxyl radical scavenging activity was measured by Haber-Weiss's catalytic ion method. Hydroxyl radicals were generated using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap and analyzed using electron spin resonance. The hydrolyzate (0.2 ml) of each enzyme was mixed with 0.2 ml of DMPO (0.3 M), 0.2 ml of FeSO ₄ (10 mM) and 0.2 ml of H ₂ O ₂ (10 mM) in phosphate buffer 100 μl was transferred to a Teflon capillary tube and measured 2 minutes and 30 seconds later using an electron spin resonance apparatus. (Measurement condition: central field, 3475 g; modulation frequency, 100 kHz; modulation amplitude, 2 g; microwave power, 1 mW; gain 6.3 x 10 ⁵ and temperature,

C) Analysis of Alkyl radical scavenging activity: Alkyl radicals were generated by AAPH treatment. After 10 mmol / L AAPH 10 mmol / L 4-POBN and a suitable concentration of the sample were mixed with phosphate buffered saline (PBS, pH 7.4), the reaction was carried out at 37 ° C for 30 minutes in a constant temperature water bath. Then, 100 ul was transferred to a Telflon capillary tube Were used for ESR spectrophotometer analysis. (Measurement condition: central field, 3475 g; modulation frequency, 100 kHz; modulation amplitude, 2 g; microwave power, 10 mW; gain, 6.3 × 10 ⁵ ;

D) Superoxide radical scavenging activity: Superoxide radicals were measured by ultraviolet irradiated riboflavin / EDTA system. The samples were incubated for 1 min at 365 nm in ultraviolet light and 0.3 mM riboflavin, 5.0 mM EDTA, and 0.1M DMPO were mixed and reacted. The reactants were transferred to a Telflon capillary tube and used for ESR spectrophotometer analysis. (Measurement condition: central field, 3475 g; modulation frequency, 100 kHz; modulation amplitude, 2 g; microwave power, 4 mW; gain, 6.3 x 10 ⁵ ;

4. Formulation of optimal antler material to minimize the loss of effective ingredient of antler and the ratio of sub ingredient to improve functionality were established.

II. result

1. Manufacture of food ingredients (deer antler and deer antler) using antler

(1) general components of elk antler

Table 1 shows the results of nutrient analysis for each part of elk antler (relative, major, and lower). The building content of each site was in the range of 27 ~ 38%. Crude protein, crude fat and total sugar content were lower in order of relative, major, and inferior. On the other hand, in the case of ash, the lowest value was 37.6%. In addition, the values of collagen, calcium and phosphorus increased as the ash increased. On the other hand, the contents of Glycosaminoglycan, uronc acid and sialic acid, which are known as physiologically active substances of antler, show a marked increase.

Generic component of elk antler Dry matter Crude protein Crude fat Ash Total carbohydrate GAGs (mg / mL) Upper 27.7 ± 1.6 51.3 ± 77.8 2.82 ± 0.4 26.09 ± 4.4 10.5 ± 1.3 2.1 ± 0.2 Middle 32.8 ± 2.3 33.27 ± 3.4 2.41 ± 0.4 38.55 + - 3.4 9.1 ± 1.7 1.8 ± 0.2 Base 37.9 ± 4.3 25.99 + - 2.8 2.45 ± 0.4 42.06 ± 3.4 7.8 ± 1.6 1.3 ± 0.1 Uronic
acid (mg / mL) Sialic
acid (mg / mL) Collagen (mg / mL) Ca
(g / mL) P
(g / mL) Upper 0.7 ± 0.3 0.9 ± 0.1 23.3 ± 2.4 13.1 ± 3.8 2.9 ± 1.6 Middle 0.6 ± 0.2 0.5 ± 0.1 26.2 ± 1.6 14.6 ± 2.5 6.4 ± 1.2 Base 0.3 ± 0.1 0.1 ± 0.1 28.6 ± 1.4 15.9 ± 1.4 9.4 ± 1.0

(2) Amino acid composition of each part of elk antler

Table 2 and FIG. 1 show the analysis results of the amino acid composition of the antler range concentrate prepared by varying the antler part. Especially proline and glycine among the free amino acids in the antler juice showed a tendency to increase with the addition of the antler of the antler. Generally, the collagen in the antler is composed of Glycine-Proline-Hydroxyproline structure, which is consistent with the result of the collagen content in the lower part of the collagen compared to the other collagen.

Analysis of Amino Acid Contents of Deer Antler Extract Prepared by Antler Part (mg%) Amino acid type By antler part Amino acid content (mg%) Amino acid type By antler part Amino acid content (mg%) Asp Upper 0.87 Tyr Upper 0.49 Middle 0.75 Middle 0.43 Base 0.93 Base 0.50 Glu Upper 1.33 Pro Upper 3.55 Middle 2.07 Middle 5.13 Base 1.44 Base 3.57 Lys Upper 0.16 Ala Upper 1.10 Middle 0.13 Middle 1.19 Base 0.20 Base 1.15 Trp Upper 0.61 Thr Upper 0.27 Middle 0.95 Middle 0.28 Base 0.52 Base 0.36 Phe Upper 0.33 Arg Upper 8.12 Middle 0.20 Middle 8.41 Base 0.38 Base 10.30 Leu Upper 0.39 His Upper 0.84 Middle 0.26 Middle 0.83 Base 0.39 Base 0.95 Ile Upper 0.29 Gly Upper 0.19 Middle 0.23 Middle 0.19 Base 0.32 Base 0.19 Cys2 Upper 0.07 Gln Upper 0.27 Middle 0.06 Middle 0.74 Base 0.05 Base 0.22 Met Upper 0.15 Ser Upper 0.41 Middle 0.24 Middle 0.42 Base 0.15 Base 0.48 Val Upper 0.38 Asn Upper 3.41 Middle 0.32 Middle 4.03 Base 0.44 Base 3.17

(3) Production process of protein and carbohydrate hydrolase of elk antler

In the present invention, an enzymatic hydrolysis extract of each part of elk antler was prepared using protein and carbohydrate hydrolase, and the enzymes and the hydrolysis process used in the present invention are shown in Table 3 and FIG.

The enzymatic properties used in the hydrolysis of antler in the present invention Enzyme Type Enzyme characterization Optimum conditions pH Temp () Celluclast Mono cellulase 4.5 50 Termamyl Mono amylase 6.0 60 AMG Mono amyloglucosidase 4.5 60 Ultraflo Complex -glucanase, cellulase
xylanase, pentosanase, arabinase 6.5 60 Viscozyme Complex -glucanase, cellulase
hemicellulase, zylanase, arabinase 4.5 50 Pepsin Mono protease 7.0 37 Alcalase Mono protease 8.0 50 Neutrase Mono protease 6.0 50 Flavourzyme Mono exopeptidase 7.0 50 Protamex Mono serine endoprotease 6.0 40

(4) Yield of protein and carbohydrate hydrolase extracts of elk antler

In the present invention, an enzymatic hydrolysis extract of each part of elk antler was prepared using protein and carbohydrate hydrolase, and the yield of each extract was shown in Table 4.

The extraction yields of all hydrolyzed extracts were the highest in all, followed by the order of major and minor. The yields of hydrolyzed and hydrolyzed hydrolyzed extracts were not significantly different from those of hydrolyzed hydrolyzed hydrolysates. The hydrolyzed hydrolyzate yields were 11.27 ~ 26.67%, 6.49 ~ 17.38%, and 4.52 ~ 11.28% Range. The difference in the degree of hydrolysis of the fractions of antler is believed to be due to the high relative content of crude protein and the low tendency of antler. In addition, the enzyme has an active site that binds to the substrate to convert the substrate to the product. Usually, the active site forms a gap in the outer part of the enzyme molecule, and thus has a complementary form to the structure of the substrate. The difference in the degree of hydrolysis by enzymes is considered to be due to the selective substrate specificity of these enzymes.

Yield of Hydrolysis Extract of Deer Enzyme Extraction yields  (%) Water extract Upper 33.42 Middle 31.05 Base 24.02 Ethanol extract Upper 29.98 Middle 8.41 Base 2.89 Celluclast Upper 21.77 Middle 10.03 Base 6.42 Termamyl Upper 15.59 Middle 11.72 Base 6.99 AMG Upper 18.21 Middle 8.89 Base 8.90 Ultraflo Upper 11.27 Middle 11.33 Base 11.03 Viscozyme Upper 23.71 Middle 6.49 Base 5.12 Pepsin Upper 20.81 Middle 15.95 Base 11.28 Alcalase Upper 20.85 Middle 10.90 Base 4.52 Neutrase Upper 26.67 Middle 14.57 Base 8.64 Flavourzyme Upper 19.88 Middle 12.65 Base 9.21 Protamex Upper 21.76 Middle 17.38 Base 8.86

(5) Physiologically active components of protein and carbohydrate hydrolase of elk antler

The results of physiologically active components of protein and carbohydrate hydrolase extracts of elk antler are shown in Tables 5 to 9 and FIGS. 4 to 8. The uronic acid content of the various enzymatic hydrolysates of elk antler was higher in the opponent and medium-sized group than in the lower group, and the highest value was observed in the termamyl and protamex hydrolysates of the antler group (Table 5, Fig. 4) . Sialic acid content was the highest in the relative antioxidant extract, especially in the Flavourzyme protein hydrolytic enzyme and Ultroflo carbohydrate hydrolyzate (Table 6, Fig. 5) .

Table 7 also. As shown in Fig. 6, the content of Glycosaminoglycan was higher than that of the other extracts. Especially, most of the protein hydrolytic enzyme extracts (Alcalase, Neutrase, Flavourzyme, Protamex) including hot water extract and Viscozyme extract Respectively. On the other hand, total sugar content was higher in carbohydrate hydrolyzed extract (AMG, Termamyl, Ultroflo) than protein hydrolyzed extract (Table 8, Fig. On the other hand, the content of hydrolysates of Flavourzyme and pepsin, which are protein hydrolytic enzymes, was high in medium and low. The content of collagen was increased from the other to the lower part. Especially, the contents of Cellucalst, AMG extract and protein hydrolytic enzymes Flavourzyme, Alcalase and Neutrase extracts were high in carbohydrate hydrolysis enzymes (Table 9 , Fig. 8).

The uronic acid content (mg / mL) of the hydrolyzed extract according to the hydrolysis enzyme of the antler Sample Average STDEV Viscozyme L Upper 0.027 0.005 Middle 0.021 0.011 Base 0.044 0.013 Termamy L Upper 0.050 0.005 Middle 0.163 0.022 Base 0.038 0.012 Cellucalst Upper 0.037 0.000 Middle 0.082 0.002 Base 0.023 0.006 Alcalase Upper 0.033 0.011 Middle 0.028 0.003 Base 0.020 0.003 AMG Upper 0.001 0.005 Middle 0.002 0.002 Base 0.021 0.010 Neutrase Upper 0.004 0.004 Middle 0.016 0.002 Base 0.003 0.008 Ultroflo Upper 0.015 0.004 Middle 0.001 0.000 Base 0.005 0.003 Flavourzyme Upper 0.010 0.001 Middle 0.057 0.007 Base 0.029 0.002 Water extracts Upper 0.131 0.015 Middle 0.010 0.000 Base 0.022 0.002 Pepsin Upper 0.017 0.002 Middle 0.037 0.015 Base 0.005 0.001 Protamex Upper 0.015 0.013 Middle 0.116 0.001 Base 0.032 0.003 Sonication Upper 0.022 0.002 Middle 0.021 0.004 Base 0.036 0.012

Sialic acid content (mg / mL) of hydrolyzed extract according to hydrolysis enzyme of antler Sample Average STDEV Alcalase Upper 0.364 0.007 Middle 0.216 0.006 Base 0.206 0.148 Viscozyme L Upper 0.205 0.002 Middle 0.247 0.150 Base 0.249 0.003 Termamy L Upper 0.399 0.011 Middle 0.157 0.008 Base 0.028 0.002 Neutrase Upper 0.313 0.009 Middle 0.386 0.004 Base 0.348 0.014 AMG Upper 0.505 0.013 Middle 0.080 0.002 Base 0.111 0.004 Flavourzyme Upper 0.622 0.009 Middle 0.478 0.015 Base 0.456 0.012 Ultroflo Upper 0.679 0.035 Middle 0.535 0.006 Base 0.383 0.006 Celluclast Upper 0.332 0.009 Middle 0.377 0.011 Base 0.366 0.009 Water extracts Upper 0.427 0.007 Middle 0.284 0.003 Base 0.158 0.006 Pepsin Upper 0.343 0.007 Middle 0.699 0.014 Base 0.475 0.067 Protamex Upper 0.357 0.014 Middle 0.466 0.007 Base 0.576 0.015 Sonication Upper 0.466 0.029 Middle 0.478 0.003 Base 0.377 0.016

Glycosaminoglycan content (mg / mL) of hydrolyzed extract according to hydrolysis enzyme of antler Sample Average STDEV Viscozyme L Upper 11.524 0.334 Middle 10.773 0.303 Base 2.591 0.077 Termamy L Upper 1.252 0.102 Middle 0.853 0.193 Base 0.697 0.102 Celluclast Upper 0.193 0.067 Middle 0.273 0.067 Base 5.577 0.566 AMG Upper 3.282 0.177 Middle 3.919 0.435 Base 2.190 0.235 Alcalase Upper 6.881 0.077 Middle 1.826 0.597 Base 2.443 0.101 Neutrase Upper 4.307 0.102 Middle 2.133 0.199 Base 2.781 0.192 Water extracts Upper 7.588 0.302 Middle 1.170 0.467 Base 0.603 0.145 Sonication Upper 2.176 0.617 Middle 2.048 0.353 Base 1.882 0.194 Ultroflo Upper 0.402 0.114 Middle 2.395 0.552 Base 1.170 0.084 Flavourzyme Upper 4.260 0.271 Middle 2.834 0.114 Base 2.505 0.138 Pepsin Upper 1.064 0.178 Middle 8.907 0.292 Base 2.866 0.564 Protamex Upper 9.100 0.292 Middle 6.958 0.547 Base 0.147 0.435

The total carbohydrate content of the hydrolyzed extract according to the hydrolysis enzyme of the antler (mg / mL) Sample Average STDEV AMG Upper 12.210 0.034 Middle 1.772 0.028 Base 1.889 0.019 Alcalase Upper 8.584 0.105 Middle 4.344 0.016 Base 2.854 0.167 Neutrase Upper 3.551 0.074 Middle 3.707 0.058 Base 4.736 0.019 ViscozymeL Upper 3.659 0.037 Middle 4.014 0.007 Base 3.591 0.061 TermamyL Upper 3.115 0.024 Middle 19.851 0.191 Base 1.751 0.007 Celluclast Upper 2.588 0.000 Middle 2.750 0.007 Base 2.542 0.019 Flavourzyme Upper 1.956 0.154 Middle 5.918 0.051 Base 9.461 0.117 Ultroflo Upper 1.583 0.144 Middle 4.678 0.187 Base 4.069 0.057 Sonication Upper 6.474 0.190 Middle 4.934 0.080 Base 6.156 0.133 Water extracts Upper 3.036 0.110 Middle 3.240 0.078 Base 3.190 0.105 Pepsin Upper 3.889 0.018 Middle 13.975 0.050 Base 4.545 0.005 Protamex Upper 4.171 0.028 Middle 4.159 0.039 Base 3.915 0.033

Glycosaminoglycan content (mg / mL) of hydrolyzed extract according to hydrolysis enzyme of antler Sample Average STDEV celluclast Upper 0.538 0.013 Middle 0.711 0.023 Base 1.238 0.034 AMG Upper 0.741 0.057 Middle 1.238 0.034 Base 1.125 0.026 Viscozyme L Upper 0.149 0.021 Middle 0.234 0.021 Base 0.255 0.037 Termamyl Upper 0.510 0.000 Middle 0.652 0.012 Base 1,000 0.021 Alcalase Upper 0.355 0.060 Middle 0.793 0.012 Base 0.883 0.021 Neutrase Upper 0.612 0.021 Middle 0.439 0.012 Base 0.522 0.012 Flavourzyme Upper 0.393 0.000 Middle 0.761 0.014 Base 0.894 0.000 Sonicate Upper 0.176 0.014 Middle 0.151 0.029 Base 0.235 0.029 Ultroflo Upper 0.117 0.007 Middle 0.364 0.099 Base 0.303 0.024 Water extracts Upper 0.140 0.017 Middle 0.689 0.033 Base 0.587 0.024 Pepsin Upper 0.359 0.036 Middle 0.165 0.016 Base 0.165 0.016 Protamex Upper 0.264 0.018 Middle 0.427 0.033 Base 0.369 0.042

(6) Radical scavenging activity of protein and carbohydrate hydrolase of elk antler

The antioxidative activity of hydrolyzate of antler starch was analyzed by ESR spectrophotometer. The results are shown in Table 10 and FIGS. 9 to 11. Antioxidative activities of antler starch hydrolysates were analyzed by DPPH, hydroxyl and alkyl radical scavenging activity and expressed as IC ₅₀ (mg / mL), which is a radical inhibitory concentration of 50% for each radical.

The antioxidative effect of all radicals was the highest in the relative group, and the antioxidative effect was lower in the major and the lower group. In addition, all radical scavenging activity increased in proportion to the concentration. Especially, DPPH radical scavenging activity showed antioxidant activity of protamex (0.96 mg / mL), Flavourzyme (1.28 mg / mL), alcalase (1.71 mg / mL) and termamyl (1.77 mg / mL)

DPPH is a relatively stable free radical with a deep purple color and is widely used to search for antioxidants from various natural materials. Free radicals are likely to cause aging by binding to lipids or proteins in the body. In the case of phenolic compounds, they have a strong ability to reduce or offset free radicals, which can be used as a measure to inhibit free radical aging in the body.

Hydroxyl radicals were generated in the Fe ²⁺ / H ₂ O ₂ system using DMPO trap. A typical 1: 2: 2: 1 ESR signal of DMPO-OH was observed. Antioxidant efficacy against hydroxyl radicals was similar to DPPH radical scavenging activity. Flavourzyme (0.89 mg / mL), protamex (1.37 mg / mL), pepsin (1.63 mg / mL), celluclast hydroxyl radical scavenging activity was higher in the order of viscozyme (1.95 mg / mL).

Radical scavenging of the hydrolysis extract according to the hydrolytic activity of the antler IC ₅₀ (mg / mL) Sample DPPH Hydroxyl Alkyl Alcalase Upper 1.71 2.76 0.46 Middle 2.64 3.11 1.42 Base 4.02 5.40 1.85 AMG Upper 2.79 2.18 2.57 Middle 5.22 2.76 3.73 Base 6.71 3.10 4.41 Celluclast Upper 3.02 1.95 2.23 Middle 4.40 2.06 2.94 Base 5.00 2.56 3.75 Flavourzyme Upper 1.28 0.89 1.64 Middle 2.34 2.12 1.83 Base 2.65 3.54 3.74 Neutrase Upper 2.24 2.43 1.55 Middle 3.56 3.18 1.74 Base 4.30 5.57 1.91 Pepsin Upper 2.06 1.63 0.42 Middle 2.95 3.42 1.37 Base 4.16 4.57 2.27 Protamex Upper 0.96 1.37 0.37 Middle 1.76 2.37 1.27 Base 3.90 2.97 1.38 Sonication Upper 2.88 3.76 3.39 Middle 3.37 4.66 4.25 Base 3.41 5.88 4.98 Termamyl Upper 1.77 2.45 1.34 Middle 2.80 2.51 3.13 Base 4.16 3.95 3.16 Ultroflo Upper 3.30 1.98 1.38 Middle 3.07 2.27 1.80 Base 3.67 4.26 2.13 Viscozyme Upper 2.86 1.95 0.79 Middle 3.51 2.28 1.18 Base 4.44 3.94 1.41 Hot water extract Upper 3.21 2.39 2.08 Middle 4.16 3.54 3.63 Base 6.37 5.97 5.11

The Alkyl radical was measured from the 4-POBN / freezer treated with AAPH for 30 min at 37 < 0 > C. In the case of Alkyl radical scavenging activity, the relative activity of the antler protein hydrolyzate was the most active and the activity was decreased as the activity decreased. The alkyl radical scavenging activities of enzyme extracts were similar to those of protamex (0.37 mg / mL), pepsin (0.42 mg / mL), alcalase (0.46 mg / mL), viscozyme (0.79 mg / mL) , Neutrase (1.55 mg / mL) and Flavourzyme (1.64 mg / mL) were superior in antioxidant activity.

These results suggest that the antioxidant capacity of the antler protein hydrolyzate may be different depending on the type of peptide produced by the selective cleavage of the enzyme to the protein. The antioxidant capacity of the protein hydrolyzate varies depending on the characteristics of the constituent peptide, which is considered to be related to the change in the stereostructure and the molecular weight of the peptide due to the terminal amino acid of the peptide produced by hydrolysis.

2. Formulation of the best antler material to minimize the loss of effective ingredient of antler and establishment of the ratio of additives to improve functionality

The results of analyzing the DPPH radical scavenging activity of the plant extract for use as an additive in the present invention are shown in FIG. In other words, in order to utilize in the development of various processed products using antler, the present invention selects ten kinds of herbal medicines known to have high antioxidative properties with reference to each kind of literature, and then, in order to measure the antioxidative activity of each plant extract, ) spectrometer was used to measure DPPH radical scavenging activity. IC ₅₀ values for the DPPH radical of the result, each plant extract, compare the respective IC ₅₀ (mg / ml) value by measuring the DPPH radical scavenging activity showed a 0.12 ~ 3.87 mg / ml range, decursiva of the The highest antioxidant activity was obtained with IC ₅₀ value of 0.12 mg / ml. On the other hand, the IC ₅₀ value of Hwanggi was 3.87 mg / ml, which showed the lowest antioxidant ability. Jujube showed almost no antioxidant ability.

Based on the results of the antioxidative activity measurement of the above plant extracts, examples of mixing ratios for developing a functional product using the enzymatic hydrolysis extract of Deer antler are shown in Table 11, and a plant mixed extract was prepared based on the results. Specific examples are as follows.

Mixing ratio of plant extract No. Types of plant extracts Blending ratio (% by weight) IC ₅₀ (mg / ml) One Ginseng 3.4 (14.9) 3.40 2 Boiled 3.45 (15.1) 3.45 3 Baekbokryung 3.47 (15.2) 3.47 4 Angelica 0.96 (4.2) 0.96 5 Celestial 0.57 (2.5) 0.57 6 Sookmyung 1.79 (7.8) 1.79 7 A black protein 0.12 (0.5) 0.12 8 Hwanggi 3.87 (16.9) 3.87 9 Broiler 3.8 (16.6) 3.80 10 licorice 1.42 (6.2) 1.42

≪ Example 1 >

1) ginseng 14.9% by weight, white 15.1% by weight, white bell 15.2% by weight, Angelica gigantis 4.2% by weight, Angelica gigantis 2.5% by weight, %, Then mix the plant mixture with an equal volume of water.

2) boil for 2 to 3 hours at 85 to concentrate and extract until the solid content reaches 15% by weight or more to prepare plant mixed extract.

3) The plant mixture extract is prepared in powder form by concentrating (liquid) or freeze-drying according to the formulation of the product to be extracted.

≪ Example 2 >

Example of mixing ratio of hydrolysis extract and sub ingredient of antler Raw material name content Remarks Hydrolyzed extract of antler
(Liquid or freeze-dried sample) 32 wt% Solid content 0.5% or more Plant extract 60.93 wt% Solid content 15% or more Grapefruit seed extract (DF-100) 0.07 wt% Other mushroom extract 7 wt% Solid content 0.3% or more Sum 100 wt%

1) A mixture of plant extracts (14.9% by weight of ginseng, 15.1% by weight of white ginseng, 15.2% by weight of white ginseng, 4.2% by weight of angelica ginseng, 2.5% by weight of ginseng root, 7.8% by weight of sorghum, 0.5% , 6.2% by weight licorice)

2) Each raw material was purchased by using suitable raw materials for food revolution and additives. A suitable raw material that was inspected was weighed to the blend ratio.

3) Ginseng 14.9% by weight, White 15.1% by weight, Baekbok 15% by weight, Angelica gangrene 4.2% by weight, Angelica giganteum 2.5% by weight, Sulfur sulfur 7.8% by weight, % Were mixed with water in a mixing extractor in accordance with the blending ratio, and concentrated and extracted until the solid content exceeded 15% by weight to prepare a plant mixed extract.

4) Plant mixture extract, hydrolyzed hydrolyzate of antler, and other mushroom extract were mixed in a mixing ratio, and the mixture was put into a concentrator and concentrated to 85% by weight to obtain a solid content of 10% by weight.

5) The grapefruit seed extract was added to the concentrate of 4), and the solid content was concentrated to 85% by weight, filtered and sterilized at 90 for 1 hour to be used as a raw material for final product development.

6) The final raw material was used as a concentrate (liquid) or lyophilized according to the formulation of the product to be developed.

The present invention relates to a method for preparing a hydrolytic enzyme-containing extract of deer antler according to the present invention, which enables to produce a deer antler extract, It can be used industrially because it can contribute to the industry.

Claims (9)

(1) mixing the mixture of green, middle, and lower green, or a mixture thereof with water at a weight ratio of 1: 4, boiling at 95 ° C for 7 hours, and concentrating under reduced pressure to obtain a deer concentration of 60 to 70 ° Brix;
(2) mixing at least one hydrolytic enzyme selected from the group consisting of Celluclast, Termamyl, AMG, Ultraflo and Viscozyme in a mass ratio of 100: 1 to the deer antler of step (1) for 24 hours;
And (3) filtering the hydrolyzate of step (2), removing the enzyme activity, and adjusting the pH to 7, followed by lyophilization, to prepare a hydrolytic enzyme-containing extract of deer antler containing active ingredient Way.
(A) preparing a hydrolytic enzyme-containing extract containing a hyperactive component of antler according to (1);
B) 16.9% by weight of white ginseng, 16.6% by weight of white ginseng, 6.2% by weight of licorice, 6.2% by weight of white ginseng, 15.1% by weight of white ginseng, 15.2% by weight of white ginseng, % Of the plant mixture is mixed with water at the same weight, and then concentrated at 85 캜 until the solid content reaches 15% by weight.
(C) mixing the hydrolytic enzyme extract of step (a) and the plant mixed extract of step (b) above to prepare a hydrolytic enzyme-containing extract of deer antler.
The hydrolytic enzyme-containing extract of deer antler produced by the method of claim 1 or 2. delete delete delete delete delete delete

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