KR20120057699A - Development of extraction technique for preparation of hydrophilic extracts and biological activities of velvet antler - Google Patents

Abstract

PURPOSE: The development of an extraction method for deer antlers for obtaining a water soluble material, and a biological activity of an obtained extract are provided to prevent the transformation of effective components inside the deer antlers after processing. CONSTITUTION: An extraction method of deer antlers for producing a water soluble material comprises a step of drying the deer antlers using far infrared rays at 1-100 deg C for 0.1-60 hours, or hot-water/enzyme extracting the deer antlers using enzymes selected from neutrase, alkalase, flavourzyme, protamax, kojizyme, protease, alpha-chymotrypsin, trypsin, papain, or pepsin. The dried deer antlers or the hot-water/enzyme extract of the deer antlers are used as an active ingredient for antihypertensive and anti-oxidative compositions, functional cosmetics, or functional food.

Description

Development of extraction technique for preparation of hydrophilic extracts and biological activities of velvet antler}

The present invention relates to the development of an extraction method for the preparation of water-soluble substances for antler and the physiological activity of the extract. To explain this in more detail, antler, which contains a large amount of collagen protein and high-quality calcium, has been the health food that Koreans have enjoyed the most along with ginseng since ancient times, but it is very difficult to extract useful ingredients from antler. There is a lack of research on functional materialization, and the situation is facing limitations in the development of products using the same. Therefore, in order to solve this problem, the present invention has been developed in a hydrothermal-enzymatic extraction method in which a pulverization method and a hydrothermal / enzyme extraction method are performed in combination with a conventional far-infrared drying method and then pulverized into a fine powder without changing the components. It relates to the biological activity of the extract.

Functional materialization using useful ingredients contained in deer antler develops the world's first functional material derived from deer antler, which has not been attempted until now, and the development of application products using it has a good opportunity to contribute to the globalization of oriental medicine. Deer antler contains a large amount of collagen protein and high-quality calcium, which is believed to contribute to the development of poor bio-material industrialization in Korea.

In addition, considering that the position of collagen peptide cosmetics in the natural high-performance cosmetics market is rapidly growing in line with the recent trend of well-being, the development of antler-derived collagen peptides has led to the development of regional and national economies with the development of functional materials market. Will be able to contribute.

Accordingly, an object of the present invention is to apply the far-infrared drying method for efficient grinding and the enzyme-thermal water extraction method for maximizing the extraction efficiency of useful components, different from the existing methods, in order to increase the extraction efficiency of useful components from deer antler for the first time in Korea and the world. It is to evaluate the physiological activity of extracts by developing effective micropowder grinding and new extraction methods and contributing to the development of domestic and overseas functional markets.

The present invention relates to the development of an extraction method for the preparation of water-soluble substances for antler and the physiological activity of the extract. To explain this in more detail, antler, which contains a large amount of collagen protein and high-quality calcium, has been the health food that Koreans have enjoyed the most along with ginseng since ancient times, but it is very difficult to extract useful ingredients from antler. There is a lack of research on functional materialization, and the situation is facing limitations in the development of products using the same. Therefore, in order to solve this problem, the present invention has been developed in a hydrothermal-enzymatic extraction method in which a pulverization method and a hydrothermal / enzyme extraction method are performed in combination with a conventional far-infrared drying method and then pulverized into a fine powder without changing the components. It relates to the biological activity of the extract.

Therefore, the technical object of the present invention is to devise to improve the various problems of the conventional extraction method presented above, the fine powder through the pulverization after far-infrared drying, in order to secure the point that the extraction efficiency is low, because the grinding efficiency is low. The collagen peptide and the natural water-soluble calcium were separated from the antler using a hydrothermal-enzymatic parallel extraction method using enzymatic hydrolysis, which is a method to compensate for the disadvantages of the existing hydrothermal extraction method. The purpose is to develop as a natural functional material by evaluating the biological activity of such hydrothermal-enzymatic extract.

Recently, as consumers' perception of the well-being boom and the functional food is spreading, interest in health functional foods and beauty materials having various physiological activities is increasing worldwide.

For example, the United States, which has the world's largest market for dietary supplements, was $ 70.7 billion in 2003, including natural organic foods, dietary supplements, dietary supplements, and natural products, about 35 out of the $ 22.3 billion globally. It has been reported to account for%. Thanks to such market size, the market reached over $ 80 billion in 2005 and is expected to continue growing by about 10% annually over the next decade.

On the other hand, in the case of Japan, the collagen market is estimated at 18.5 billion yen, and in the health food market, which is estimated at 500 billion yen, the collagen compound product, a kind of protein that is known to have a cosmetic effect such as skin moisturizing effect, is growing rapidly. We are seeing and are attracting attention.

The domestic collagen peptide industry is also growing in demand due to changes in health-oriented perceptions of consumers, and industry investment is actively being made, but research investment is still insufficient compared to the developed countries in the United States.

In addition, there is a need for the discovery and research of new natural collagen peptides mainly due to the consumer's distrust of the raw materials to obtain collagen peptides (mad cow disease, foot and mouth disease, heavy metal contamination, etc.).

In addition, the antler extract having excellent functionality has been used so far with solvent extraction methods such as hot water and ethanol extraction and physical extraction methods using ultrasonic and high pressure devices. However, not only the yield of the extracted useful physiologically active substance is low, but also the destruction of protein components and useful components such as vitamins in the extraction process, safety problems resulting from the use of organic solvents, and expensive equipment installation problems have been reported.

In addition, most of the antler-related products in Korea are hot water extracted in the form of mixed antler and various herbal medicines, and the extracts are sold in pouch form, and there are no applications and studies on collagen peptides due to the above problems. .

As such, in the development of the antler-related products, the application of the existing hot water extraction method or organic solvent extraction method is difficult to meet the expected effects when using the antler extract as it easily destroys the bioactive components or causes a problem in safety. Therefore, there is a demand for the development of a method for extracting an active ingredient having secured safety while reducing the damage of the physiologically active substance.

One way to solve the problem is enzymatic extraction. Enzymatic extraction method can easily obtain useful materials by extracting smaller units of water-soluble substances, the process is also easy and the yield is higher than the existing method is very efficient when applied to the food industry. Therefore, the hydrothermal-enzymatic parallel extraction method using the enzymatic extraction method is a very efficient method to achieve the maximum yield.

In addition, extraction using hydrothermal-enzymatic parallel extraction? When antler-derived collagen peptides have been purified and functional, such as antihypertensive (angiotensin I-converting enzyme inhibitory activity), antioxidant, anti-aging, whitening, and UV-blocking effects, they are likely to be of considerable industrial potential. Natural materials can contribute to economic development.

In particular, functional materials utilizing the useful ingredients contained in deer antler are developing the world's first functional deer antler-derived functional material, which has not been attempted until now. Deer antler contains a large amount of collagen protein and high-quality calcium, which is believed to contribute to the development of poor bio-material industrialization in Korea.

In addition, considering that the position of collagen peptide cosmetics in the natural high-performance cosmetics market is rapidly growing in line with the recent trend of well-being, the development of antler-derived collagen peptides has led to the development of regional and national economies with the development of functional materials market. Will be able to contribute.

Accordingly, an object of the present invention is to apply the far-infrared drying method for efficient grinding and the enzyme-thermal water extraction method for maximizing the extraction efficiency of useful components, different from the existing methods, in order to increase the extraction efficiency of useful components from deer antler for the first time in Korea and the world. It is to evaluate the physiological activity of extracts by developing effective micropowder grinding and new extraction methods and contributing to the development of domestic and overseas functional markets.

As described above, the present invention improved the extraction efficiency by pulverizing antler, which has been difficult to grind up to now, after far-infrared drying, and finely powdered by pulverization. It has shown its potential as calcium.

Figure 1. Flowchart of hydrothermal-enzymatic hydrolysis process of antler
Figure 2. Comparison after crushing according to antler far infrared drying method and other drying method
Figure 3. Yield and hydrolysis of antler extract by enzyme
4. Angiotensin I-converting enzyme inhibitory activity according to antler hydrothermal-enzymatic extraction method
5. Hydrolysis degree according to the substrate concentration of antler hydrothermal-alkalase hydrolysate
Figure 6. Hydrolysis according to substrate vs. enzyme of antler hydrothermal-alkalase hydrolysate
7. Antihypertensive activity according to antler hydrothermal-alkalase hydrolyzate concentration
8. Analysis of general components of antler hydrothermal-alkalase hydrolyzate

The present invention relates to the development of an extraction method for the preparation of water-soluble substances for antler and the physiological activity of the extract. To explain this in more detail, antler, which contains a large amount of collagen protein and high-quality calcium, has been the health food that Koreans have enjoyed the most along with ginseng since ancient times, but it is very difficult to extract useful ingredients from antler. There is a lack of research on functional materialization, and the situation is facing limitations in the development of products using the same. Therefore, in order to solve this problem, the present invention has been developed in a hydrothermal-enzymatic extraction method in which a pulverization method and a hydrothermal / enzyme extraction method are performed in combination with a conventional far-infrared drying method and then pulverized into a fine powder without changing the components. It relates to the biological activity of the extract.

Example 1 Preparation of Materials

Deer antler used in this experiment was stored at -40 ° C until it was crushed after it was purchased at Jinyong Sam Deer Farm.

Example 2 Establishment of Optimal Grinding Conditions for Deer Antlers

To increase the yield of the extract was compared by drying the antler not easy to grind by temperature, time using high temperature drying, freeze drying, far-infrared drying. In case of high temperature drying, 6 hours were dried at 60 ° C. and 6 hours at 80 ° C., respectively, and lyophilized was dried at −50 ° C. for 24 hours. Finally, in the case of far-infrared drying, drying was performed at 60 ° C. for 6 hours and at 80 ° C. for 6 hours, respectively. The dried antler was ground using a grinder (KNIFTEC 1095, Sample Mill) into particles that can pass through a 0.1 mm sieve and stored frozen at -40 ℃ until chemical analysis.

[ Example  3] derived from antler Hydrothermal - Enzymatic  Extraction Method

A hydrothermal-enzymatic parallel extraction method using the enzyme extraction technique and the hydrothermal extraction technique using hydrolysis of the enzyme was used (FIG. 1). That is, 100 ml of distilled water was added to 1 g of the lyophilized sample, hot water was extracted for 24 hours, and then 10 mg of each enzyme was mixed and reacted. Enzyme hydrolysis was performed at the optimum temperature of each enzyme for 24 hours, and then the reaction was stopped by heating at 100 ° C. for 10 minutes, and then the supernatant was lyophilized by centrifugation of the hydrolyzed sample and then -40 ° C. Used while storing at. Each hydrolysis condition is as follows (Table 1).

Table 1. Optimal Conditions by Enzyme enzyme pH Temperature (℃) origin protein
Degrading enzyme Alpha Chymotrypsin 7.8 25 Bovine pancreas Alkalase 8.0 50 Bacillus licheniformis Papain 6.2 25 Carica papaya Trypsin 7.6 25 Procine pancreas

[ Experimental Example  1] Optimum Drying Method of Deer Antler

In the case of pulverizing the antler obtained through the conventional freeze-drying, there was a problem that the particle size is large and not uniform. This problem causes the problem again that the components of the extract is not constant when the antler extract. In order to solve these various problems, we searched for the conditions in which the samples were ground finely and evenly.

After the high temperature, freezing, and far-infrared drying to facilitate extraction, the grinding was performed. As a result, the method of drying using far-infrared rays compared to other drying methods (high temperature, freeze-drying) in which the size of the ground sample is not uniform and the particles are large. (80 ℃, 6hr) showed no constituent change and completely crushed (Figure 2) (Table 2).

Table 2. Other Components in the Drying Method of Antlers (unit : %) Sample Drying method Crude protein moisture Views min Etc velvet Before drying 51.1 ± 4.8 11.6 ± 1.6 35 ± 6.1 2.3 High temperature drying 53.47 ± 0.1 0.73 ± 0.25 42.1 ± 0.5 3.7 Far Infrared Drying 58.24 ± 0.1 4.9 ± 0.1 34.5 ± 0.3 2.36

[ Experimental Example  2] Analysis of general components of antler

Table 3. Common Components of Antlers (unit : %) Sample Crude protein moisture Views min Etc velvet 58.24 ± 0.1 4.9 ± 0.1 34.5 ± 0.3 2.36

Table 3 shows the results of the analysis of the general components of antler based on the method of AOAC (1990). Crude protein (58.24%) and crude ash (34.5%) were significantly higher, while other components were relatively low.

[ Experimental Example  3] Enzymatic  Hydrolysis of Deer Antler by Method

After hydrothermal extraction to hydrolyze the dried and powdered antler, four proteolytic enzymes (alphachymotrypsin, alkalase, papain and trypsin) were used to measure the degree of hydrolysis according to the hydrolysis reaction time. In addition, the hydrolysis degree was measured according to the hydrolysis reaction time by using four enzymes without hot water extraction process. As a result, the hydrolysis degree of hydrolyzate extracted by the hydrothermal-enzymatic extraction method and the hydrolysis degree hydrolyzed using only enzyme showed a sharp increase in degree of hydrolysis up to 12 hours. It was almost constant. However, as a result of comparing the degree of hydrolysis according to the extraction method, it was confirmed that the hydrothermal-enzymatic hydrolysis showed higher yield and degree of hydrolysis. In addition, among the four types of hydrolyzate, the yield of alkalase hydrolyzate was about 50%, but the alpha chymotrypsin was most effectively hydrolyzed in the degree of hydrolysis, and about 12 hours after the hydrolysis reaction. Hydrolysis degree of more than 60% was shown (Fig. 3).

[ Experimental Example  4] Antler Collagen Hydrolyzate Angiotensin  I-transferase inhibitory activity

Determination of ACE Inhibition with Deer Antler-Derived Collagen Hydrolysates Prepared Using Four Different Proteinases (Alphachymotrypsin, Alkalase, Papain and Trypsin) to Measure Antihypertensive Activity of Deer Antler Collagen Hydrolysates It was measured according to the method of Cushman and Cheung (1971). 50 μL of 25 mU / mL ACE enzyme solution was added to 50 μL of the collagen hydrolyzate extracted by the four enzymes, and then incubated at 37 ° C. for 10 minutes. 100 LL of 12.5 mM Hip-His-Leu (HHL) solution was added thereto as a substrate and reacted at 37 ° C. for 60 minutes, and 0.25 mL of 1N HCl solution was added thereto, followed by stirring with a test tube mixer to stop the reaction. Ethyl acetate 350 LL was added to the reaction solution, the mixture was stirred, and centrifuged (4000 rpm, 10 min) to obtain 200 LL of the supernatant (ethyl acetate layer). The aliquot was completely dried in an 80 ° C. dryer, dissolved in 1 mL of distilled water, and absorbed at 228 nm to show the ACE inhibition rate of the collagen hydrolyzate of each enzyme (FIG. 4).

% Inhibition of ACE = [1- (S-Sc) / (B-Bc)] × 100

S = absorbance measured for each sample

Sc = absorbance of each sample without ACE enzyme and reaction time 0

B = absorbance measured without sample

Bc = absorbance at zero reaction time without ACE enzyme and sample

Deer Antler-derived collagen hydrolysates prepared according to various enzymes and hydrolysis times clearly inhibited angiotensin I-converting enzyme inhibitory activity. In particular, hydrothermal-alkalase hydrolyzate showed high efficiency in terms of angiotensin I-convertase inhibitory activity and industrial application. Angiotensin I-converting enzyme inhibition pattern of antler collagen-derived hydrolysates showed a close correlation with hydrolysis time as well as antioxidant activity. In view of these results, it is judged that antler has various physiological activities only when it is decomposed to below a certain molecular weight.

[ Experimental Example  5] Antler Hydrothermal - Alkalase  Collagen Hydrolyzate  Optimum hydrolysis conditions Angiotensin  I-transferase inhibitory activity

In order to determine the optimal hydrolysis conditions of the alkalase collagen hydrolyzate showing the highest angiotensin I-converting enzyme inhibitory activity, experiments were performed under various conditions depending on substrate-to-enzyme ratio and substrate concentration. First, the degree of hydrolysis was measured using an alkalase collagen hydrolyzate with a substrate-to-enzyme ratio of 10, 20, 50, 100, 200, 500 (wt / wt). As shown in FIG. As the ratio of enzyme decreased, that is, as the concentration of enzyme increased, the degree of hydrolysis was higher. In addition, as the ratio of collagen to enzyme was increased from 10 to 100 (wt / wt), the degree of hydrolysis was higher than 50%, and from 200 (wt / wt), it was confirmed that the degree of hydrolysis rapidly dropped. As a result, it was confirmed that among the various substrate-to-enzyme ratios, substrate-to-enzyme ratio 100 (wt / wt) showed the highest efficiency for hydrolysis degree.

Therefore, the following experiment was carried out by adjusting the concentration of the substrate antler derived collagen at 1%, 3%, 5%, 10%, 20% at 100 (wt / wt). As a result, as shown in FIG. 6, the lower the concentration of the substrate, the higher the degree of hydrolysis. However, when the substrate concentration was 5% or less, the degree of hydrolysis was almost the same. do.

From the results thus far, hydrolysis was performed under various conditions depending on time, substrate-to-enzyme ratio, and substrate amount using various enzymes from collagen derived from antler. From this, the highest degree of hydrolysis was confirmed when hydrolyzed using alkalase at pH 8.0, 50 ° C, 12 hr, substrate-to-enzyme ratio of 100 (wt / wt) and substrate concentration of 5%. It was confirmed that the optimum conditions for the preparation of antler-derived collagen hydrolyzate.

Collagen hydrolyzate prepared under the optimal hydrolysis conditions of antler-derived collagen was measured angiotensin I-convertase inhibitory activity at various concentrations (0.25, 0.5 and 1 mg / mL). The activity was inhibited by about 60%, and its IC ₅₀ value was found to be 0.78 mg / mL (FIG. 7).

From these results, it was confirmed that the cleavage site of the protein is more important than the resolution in the angiotensin I-convertase inhibitory activity. In addition, it was confirmed that the antler-derived collagen hydrolyzate may have value as a raw material of a new antihypertensive agent by having an ACE inhibitory activity.

[ Experimental Example  6] Antler Hydrothermal - Alkalase Hydrolyzate  General ingredient measurement

By hydrothermal-enzymatic extraction In order to determine the accurate general composition of extracted antler-derived collagen, Konkuk University Bio Formula? As a result of confirming the content of a total of nine general components by requesting to the pharmaceutical research center, it was confirmed that the antler-derived collagen was about 94%, and most of the protein content (FIG. 8).

Figure 1. Flowchart of hydrothermal-enzymatic hydrolysis process of antler
Figure 2. Comparison after crushing according to antler far infrared drying method and other drying method
Figure 3. Yield and hydrolysis of antler extract by enzyme
4. Angiotensin I-converting enzyme inhibitory activity according to antler hydrothermal-enzymatic extraction method
5. Hydrolysis degree according to the substrate concentration of antler hydrothermal-alkalase hydrolysate
Figure 6. Hydrolysis according to substrate vs. enzyme of antler hydrothermal-alkalase hydrolysate
7. Antihypertensive activity according to antler hydrothermal-alkalase hydrolyzate concentration
8. Analysis of general components of antler hydrothermal-alkalase hydrolyzate

Claims (9)

   How to dry antler with far infrared rays    The method of claim 1, wherein the method of drying in the far infrared is a method of drying the antler at 1 to 100 ° C.     The method of claim 1, wherein the method of drying in far infrared rays is a method of drying antler for 0.1 to 60 hours. The method of claim 1, wherein the method of drying in far-infrared is a method of drying an antler for 0.1 to 60 hours at 1 to 100 ° C.
 Deer antler produced by the method of any one of claims 1 to 4. Antihypertensive, antioxidant composition, functional cosmetics or functional foods comprising the antler of claim 5 as an active ingredient Hydrothermal extract of Deer Antler was hydrolyzed with one or more enzymes selected from the group consisting of Neutrase, Alcalase, Flavozyme, Protamax, Cozyzyme, Protease, Alpha Chymotrypsin, Trypsin, Papain and Pepsin Method for preparing hot water-enzyme extract of deer antler Hot water-enzyme extract of antler prepared by the manufacturing method of claim 7
Antihypertensive, antioxidant composition, functional cosmetic or functional food comprising the hot water-enzyme extract of antler of claim 8 as an active ingredient

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