KR20130087670A - Method for preparing antler extract containing increased sialic acid content - Google Patents

Abstract

PURPOSE: A production method of a deer antler extract is provided to separate the high contents of sialic acid contained in deer antlers by high-pressure homogenization and enzyme reaction. CONSTITUTION: Deer antlers are mixed with 5-15 times the amount of water, and the mixture is heated to obtain an extract. Proteinase is added to the extract to obtain an enzyme decomposed product. The enzyme decomposed product is high-pressure homogenized. Bacteria are selected from a group consisting of Bacillus subtilis, Bacilus licheniformis, and Rhizopus. 0.1-5wt% of proteinase is added to the deer antlers. The high-pressure homogenization is conducted in a pressure of 500-1,000 bar for 1-5 times. The deer antlers after the high-pressure homogenization passes through an activated carbon packed tower for purifying. [Reference numerals] (AA) Deer antlers + purified water; (BB) Heat-processing; (CC) Enzyme reaction; (DD) Deactivating and filtering; (EE) High-pressure homogenizing; (FF) Decompress-concentrating; (GG) Drying; (HH) High-pressure homogenized deer antler extract

Description

Method for preparing antler extract with increased sialic acid content {Method for preparing antler extract containing increased sialic acid content}

The present invention relates to a method for producing an antler extract with an increased sialic acid content from an antler, and to a method for producing an antler extract having a high content of sialic acid by treating proteolytic enzymes with high pressure homogenization. will be.

Deer antler (Cervi Parvum Corun, Antler) usually refers to a deer's horn, a male's horn that is slightly soft and covered with hair and is soft enough to touch if not yet ossified within two months of its growth. In the fall, the horns that faltered hardened are keratinized, which is a fight for the females during the estrous period. These keratinized horns are called rusted and less effective than antlers. In addition, they lose their harvesting time, become calcium, and harden to fall on their own.

Deer antler contains sialic acid, hexose, pentose, hexosamine, and uronic acid, and free amino acids and prostaglandins, a type of special fatty acid widely distributed in minerals and animal tissues. It has been reported that gangliosides, a kind of leucolipids and glycolipids, exist. In particular, gangliosides, one of the complex glycolipids, have a polar head that combines several sugar units and contain one or more sialic acids.

The sialic acid is a generic term for acyl derivatives of neuramic acid (neuraminic acid), and more than 20 species are currently known in the animal kingdom. Receptor action against viruses, neutralization of toxins, immunological recognition sites of cells and molecules, cell adhesion or cancer It is reported to be an acidic sugar that is involved in important physiological functions such as metastasis. It is only present temporarily in metabolism, but mostly in the form of oligosaccharides by being glycosided at the non-reducing end of the sugar chain.

According to the literature such as Dongbobogam, deer antler has been recorded to have tonic action, blood donation, gangjeong action, analgesic action, hematopoietic action, growth growth promotion action, heart failure treatment action and hyperactivity action, in addition to fatigue recovery, physical vitality It is known to have a lot of efficacy, such as strengthening and diuretic function of the kidney.

The traditional method of taking antler used in oriental medicine is to mix and crush antler with various kinds of herbal medicines, and then heat extract with water as a solvent to take the filtrate. How to take is mainly used. Each of the two methods has advantages and disadvantages. When the extract is taken, the useful ingredient of the antler is easy to absorb while the useful ingredient contained in the extract foil is discarded. In addition, when the antler extracted and concentrated to be used, there is a problem of gelation when the solid content is 20% or more, so it is difficult to apply to the product. 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.

On the other hand, Patent Document 1 (Korean Patent Publication No. 2003-0073134; Deer Antler Extract and Extraction Method thereof) discloses a Deer Antler extraction method in which water is added to fine antler, heated and extracted under reduced pressure, and the filtrate is concentrated and spray dried to powder. Although, to improve some of the hot water extraction method used in traditional herbal medicine and to make the extract powder easy to take, there is a problem that a significant amount of the active ingredient contained in the extraction foil is not used and discarded.

Patent Document 2 (Korean Patent Publication No. 2005-0090041; Method of extracting active ingredient from antler or rust) is to extract antler or rusted pulverized product sequentially by hot water extraction, extraction using protease and hydrochloric acid extraction method. Or it relates to a method of extracting the active ingredient of green rust in high yield, but this method is complicated and time-consuming as the process proceeds to several steps, there is a problem that the use of strong acid destroys the bioactive substances useful to the human body .

Patent document 3 (Korean Patent Registration No. 1039887; strain of Cordyceps genus having activity in antler fermentation and method for producing antler fermentation extract using the same) is Cordyceps Militaris having activity in antler fermentation militaris ) KCTC 11455BP and a method for producing a deer antler fermentation extract using the strain indicates that the preparation of the deer antler fermentation extract containing a high content of sialic acid in the prior art.

Patent document 4 (Korean Laid-open Patent Publication No. 2011-0009521; strain of Bacillus genus having activity in antler fermentation and a method of producing antler fermentation extract using same) is Bacillus subtilis ( Bacillus subtilis) strain which is active in antler fermentation. subtilis ) As a method of preparing an antler fermentation extract using KCTC 11454BP, an antler fermentation extract containing a high content of sialic acid is proposed.

Patent Document 5 (Korean Patent Registration No. 1000886; Method of Manufacturing Antler Extract) adds and mixes antler and water at a predetermined ratio, and places a jar containing a mixture of antler and water in a high pressure bath in a high pressure bath. The present invention relates to a method of extracting high purity antler extract by decoction in a boiling water (中 湯) without moisture evaporation for a predetermined time under conditions.

However, these prior art obtains the antler extract through a process such as heat treatment, enzymatic decomposition, hydrochloric acid decomposition to increase the content of antler, but does not present a specific technique for increasing or separating the content of the active ingredient. Therefore, there is a need for the development of a new method for producing an antler extract containing a high content of the active ingredient of the antler while improving the problems of the conventional method of manufacturing an antler extract as described above.

Korean Patent Publication No. 2003-0073134 Korean Patent Publication No. 2005-0090041 Korean Registered Patent No. 1039887 Korean Patent Publication No. 2011-0009521 Korean Patent Registration No. 1000886

An object of the present invention is to improve the problems in the conventional manufacturing method of the antler extract as described above, to increase the extraction yield through the enzyme reaction using a protease in antler and high pressure homogenization treatment to the high content of sialic acid contained in the antler It is to provide a method for producing a antler extract with an increased sialic acid content by separation.

In order to achieve the above object,

Adding purified water to antler and heating to obtain an extract;

Adding proteolytic enzyme to the extract to obtain an enzymatic digest; And

It provides a method for producing an antler extract with increased sialic acid content comprising the step of high pressure homogenization of the enzymatic digest.

In the above method, the antler may be taken directly from a deer or commercially available. Deer antler may be used, but not limited to live antler, heat-dried antler, lyophilized antler, and can be prepared in the form of crushed antler by washing the antler clean, removing water and freezing and then crushed. As the crushing method of the antler, a method such as cutting, slicing or grinding can be used.

The crushed antler prepared as described above will heat-treat the antler extract by a hot water extraction method. In order to heat-treat the antler, 5 to 15 times the weight of the antler, preferably 10 times the purified water, is added to the crushed antler, and at 95 to 105 캜, preferably 100 캜, for 1 to 10 hours, preferably 1 to It may be heated for 3 hours, but is not limited thereto.

The protease added to the extract obtained by heating as described above is an enzyme that catalyzes the hydrolysis of peptide bonds in a protein or peptide, and any enzyme having proteolytic activity can be used, and it is a protease obtained from bacteria. Preferably, the protease obtained from the bacterium is Bacillus subtilis ( Bacillus subtilis ), Bacilus licheniformis or Rhizopus sp. is more preferably a protease obtained from any one bacterium selected from the group consisting of, and most preferably a protease obtained from Bacillus subtilis. Preferred but not limited to.

The protease is preferably added in an amount of 0.1 to 5% by weight based on the weight of the antler and reacted at 30 to 65 ° C for 3 to 15 hours, and 40 to 60 for 5 to 15 hours by adding 1 to 5% by weight. More preferably, the reaction is carried out at 55 ° C., and the reaction is performed at 55 ° C. for 5 hours by adding 1% by weight, but is not limited thereto.

In the above method, the high pressure homogenization can be carried out using a high pressure homogenizer, it is preferably carried out 1 to 5 times at 500 to 1,500 bar pressure, more preferably carried out three times at 1,000 bar pressure. By high pressure homogenization of the antler extract, sialic acid, which is a component of the ganglioside included in the antler, may be contained in the extract in a high content.

The method may further include the step of removing the color and smell of the antler by purifying the antler extract after passing the high pressure homogenization step through the activated carbon filling tower.

According to a preferred embodiment of the present invention, the pulverized antler is added to 5 to 15 times, preferably 10 times, water based on the weight of the antler to obtain an extract. To the extract obtained by heating, a protease derived from Bacillus subtilis, which is a protease, is added in an amount of 1 to 5% by weight based on the weight of antler to induce an enzyme reaction at 55 ° C. for 5 to 15 hours. Then, the enzyme reactant is homogenized at 500 to 1,000 bar pressure using a high pressure homogenizer, and the number of high pressure homogenizations is performed 1 to 5 times, preferably 3 times (see FIG. 1).

According to another preferred embodiment of the present invention, the antler extract prepared as described above may be purified by passing through an activated carbon packed tower filled with activated carbon to decolorized and deodorized purified antler extract. When preparing an extract using the present antler, when extracting a single antler and concentrated or powdered antler, there was a problem that it is difficult to use for products such as liquid in the product formulation due to the smell of antler unique in sensory sense. By removing the odor, color and impurities of the antler extract through the purification step it can be effectively used as an active ingredient of health functional food or cosmetics.

The antler extract prepared by the above method increases the extraction yield and the content of useful components in the extract according to the above enzyme treatment and enzyme reaction time in the treatment of proteolytic enzymes. 5-15 hours of treatment at 1 or 5% by weight of the extract, the extraction yield is 40% or more (see Table 1), and the collagen or sialic acid content in the antler extract is significantly increased (see Tables 2 and 3). . Considering the increase in the extraction yield and the sialic acid content of the antler extract, the enzyme treatment amount is 1% by weight based on the weight of the antler, and the enzyme reaction time is most preferably 5 hours.

The proteolytic enzyme-treated enzymatically decomposed sialic acid content is specifically increased by high pressure homogenization, and the sialic acid content is significantly increased by repeating the high pressure homogenization three times or more at 1,000 bar pressure (Table 4 and 5). In addition, the antler extract of the present invention prepared as described above exhibits excellent sensory properties compared to the high pressure homogenization and activated carbon tower untreated group by a series of proteolytic enzyme treatment, high pressure homogenization treatment and activated carbon tower treatment (see Table 6). ).

Therefore, as the deer antler extract was prepared through heat treatment, proteolytic enzyme treatment, and high pressure homogenization of the antler, the extraction yield and the content of sialic acid were increased, and a sensory excellent antler extract was obtained. Silver may be usefully used to prepare an antler extract with a specific increase in sialic acid content.

The present invention also provides an antler extract with an increased sialic acid content prepared by the above method.

As the antler extract is prepared by the preparation method as described above, the sialic acid content in the extract is preferably 0.15 to 0.35% by weight, more preferably 0.2 to 0.3% by weight, but is not limited thereto.

The antler extract prepared by the production method of the present invention increases the extraction yield and useful ingredient content by treating proteolytic enzymes in the preparation of the extract, and specifically increases the sialic acid content according to the high pressure homogenization treatment.

Therefore, the antler extract prepared in the present invention may be usefully used as an antler extract containing a large amount of sialic acid, as well as a high yield of sialic acid.

The preparation method of the antler extract according to the present invention overcomes the conventional problems of complex process, destruction of effective bioactive components and low organoleptic properties in preparing the antler extract, and the yield and useful components of the antler extract by using proteolytic enzymes. Increasing the extraction of the lysate, high sialic acid can be prepared by the high-pressure homogenization treatment of the enzyme digestion.

1 is a view showing a manufacturing process of the antler extract with an increased sialic acid content.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

< Example  1> Establishment of Enzyme Treatment Conditions of Deer Antler and Preparation of Deer Antler Extract

<1-1> Extraction yield confirmation according to enzyme reaction time and enzyme throughput

After crushing the antler purchased in the market (Gyeongdong Market) and put the crushed antler 100 g 1,000 ml of water and sterilized by heat treatment at 100 ℃ for 1 hour. 0.1, 0.5, 1, and 5 g of Bacillus subtilis containing a protease bacterial protease were respectively added to the sterilized antler aqueous solution and reacted at 55 ° C. for 1, 3, 5, 10 or 15 hours. Thereafter, 50 ml of each reaction solution was collected, and 10 g of perlite as a filter aid was added to each enzyme reaction solution, and the resultant was filtered under reduced pressure to measure solid content of the filtrate to measure extraction yield according to the reaction time and the amount of enzyme treatment.

As a result, as shown in Table 1, the extraction yield tended to increase as the enzyme throughput increased, but the extraction yield increased to 42-43% and did not increase any more. In addition, when the amount of enzyme treatment was 1 g or more, the extraction yield of 40% or more appeared in 5 hours, and thereafter, the extraction yield did not increase.

Comparison of Extraction Yields from Raw Materials According to Enzyme Reaction Time and Enzyme Processing enzyme
Throughput (g) Enzyme reaction time 1 hours 3 hours 5 hours 10 hours 15 hours 0.1 11.4% 13.1% 15.3% 20.9% 21.1% 0.5 12.1% 16.7% 20.4% 35.4% 41.6% One 16.3% 24.7% 42.1% 43.6% 42.4% 5 22.1% 36.4% 43.1% 42.9% 43.8%

<1-2> Collagen Content Identification of Antler Extract According to Enzyme Reaction Time and Enzyme Treatment

In order to measure the content of collagen which is a useful ingredient in each antler extract prepared in Example <1-1>, it was calculated by measuring the content of hydroxyproline. Specifically, each antler extract sample was taken and hydrolyzed by 6 M-HCl at 130 ° C. for 6 hours, diluted with NaOH, diluted appropriately with distilled water, and chloramine-T using a hydrolysis solution. After adding (chloramine-T) to the solution for a certain time, 4-dimethylamino benzaldehyde (4-dimethylamino benzaldehyde) was added to the reaction. After measuring the absorbance of the solution reacted with the color developing reagent at 550 nm, the absorbance was substituted into the calibration curve obtained from the hydroxyproline standard curve to measure the concentration.

As a result, it was found that the collagen content in the extract increased as the enzyme treatment time increased.However, when the enzyme treatment amount was 1 g or more, the collagen content in the extract appeared to be 8% or more in 5 hours. There was no difference (Table 2).

Collagen content in extracts according to enzyme reaction time and enzyme throughput Enzyme throughput
(g) Enzyme reaction time 1 hours 3 hours 5 hours 10 hours 15 hours 0.1 1.21% 1.42% 2.49% 3.67% 3.83% 0.5 1.34% 1.31% 2.41% 6.42% 8.16% One 1.93% 5.27% 8.11% 8.21% 8.19% 5 2.34% 6.16% 8.21% 8.23% 8.29%

<1-3> Confirmation of Sialic Acid Content in Antler Extract According to Enzyme Reaction Time and Enzyme Processing

In order to measure the content of sialic acid, which is another useful ingredient in each antler extract prepared in Example <1-1>, sialic acid fluorescence labeling kit (Sakara BIO INC.) Was used. High performance liquid chromatography (HPLC) analysis was performed by attaching a fluorescent substance to the acid.

As a result, in the case of sialic acid, as in the collagen identified in Example <1-2>, the sialic acid content was found to be 150 mg / g or more when the enzyme treatment amount was treated at 1 g or more for 5 hours or more. Increasing the abnormal enzyme treatment time did not show a significant difference in the change in content (Table 3).

Therefore, when the extraction yield and the content of the useful components according to the extraction time and the enzyme treatment amount were confirmed, it was found that the most effective when the enzyme treatment amount was 1 g and the enzyme treatment time was 5 hours.

Sialic acid content in extracts according to enzyme reaction time and enzyme throughput (mg / 100g) enzyme
Throughput Enzyme reaction time 1 hours 3 hours 5 hours 10 hours 15 hours 0.1 11.1 15.8 25.4 30.1 51.8 0.5 9.4 29.5 58.9 84.2 127.6 One 54.6 96.2 162.1 157.5 158.4 5 89.1 126.4 159.1 153.6 165.7

< Example  2> for enzyme treatment antler extract High pressure homogenization  process

On the basis of the results according to the enzyme reaction time and the enzyme treatment amount confirmed in Example 1, the enzyme treatment amount was 1%, and the enzyme was reacted for 5 hours to prepare an antler extract, followed by a high pressure homogenizer (Microfluidics, USA). Using a high pressure homogenizer (Micro fluidizer). As the high pressure homogenization conditions, the pressure was 500 or 1,000 bar, and the number of high pressure homogenizations was 0, 1, 3, and 5 times, respectively. Were analyzed as in Examples <1-2> and <1-3>, respectively. Then, the high pressure homogenized antler extract was concentrated under reduced pressure to prepare a liquid phase or dried to powder.

As a result, when the pressure was set to 500 or 1,000 bar, the content of collagen and sialic acid was compared according to the high pressure homogenization number, and when the high pressure was homogenized at a pressure of 1,000 bar, the content of sialic acid was specifically increased. The number of sialic acids increased rapidly up to three times, but there was no significant difference when the number of repetitions was increased from three to five times. However, it was found that the content of collagen did not show a large difference in content regardless of the number of high pressure homogenization (Tables 4 and 5). Therefore, it was confirmed that an effective high pressure homogenization method for preparing an antler extract having a high sialic acid content was repeated three times at 1,000 bar.

Useful ingredient content according to the number of homogenization at 500 bar Functional ingredient content High Pressure Homogenization Count 0 One 3 5 Collagen(%) 7.12 7.19 8.23 8.16 Sialic acid (mg / 100g) 153.1 164.5 186.8 201.2

Useful ingredient content according to the number of homogenization at 1000 bar Functional ingredient content High Pressure Homogenization Count 0 One 3 5 Collagen(%) 7.12 8.35 9.14 8.91 Sialic acid (mg / 100g) 153.1 191.4 248.5 251.4

< Example  3> enzyme treatment and High Pressure Emulsification  Purification and Sensory Evaluation of Deer Antler Extract

In order to remove the odor and color so that the antler extract of the present invention can be used in health functional food or cosmetics, the antler prepared in Example 2 on an activated carbon tower filled with 10-20 mesh granular activated carbon (primary carbon product) The extract was passed through to remove impurities. The concentrated solution remaining after the liquid was washed and concentrated and used in combination with the antler extract which was initially passed through. The sensory evaluation according to the number of enzyme treatment and high pressure homogenization was performed on the purified antler extract deodorized and decolorized as described above and compared with the control group (high pressure homogenization and activated carbon tower untreated group). The evaluation method is to select 30 adult men and women to explain the purpose of evaluation and to evaluate color, taste, off-flavor, and overall palatability. 5 points scale (very good 5 points, good 4 points, normal 3 points, bad 2 points, very bad 1 point), the questionnaire was evaluated, and the total scores were added and averaged.

As a result, as shown in Table 6, after the enzyme treatment and high pressure homogenization, the antler extract passed through the activated carbon tower was found to have excellent overall palatability compared to the control (Table 6).

division Homogenizer Control group 500 bar 1,000 bar flavor 4.256 4.452 4.554 4.568 4.632 4.622 2.564 Odor 4.325 4.256 4.364 4.238 4.548 4.365 2.456 Color 4.216 4.245 4.321 4.562 4.654 4.523 2.126 Overall palatability 4.365 4.354 4.368 4.346 4.564 4.652 2.246

Claims (10)

Adding purified water to antler and heating to obtain an extract;
Adding proteolytic enzyme to the extract to obtain an enzymatic digest; And
Method for producing an antler extract with increased sialic acid content comprising the step of high pressure homogenization of the enzymatic digest. The method according to claim 1,
The heating method is characterized in that for 5 to 15 times the amount of antler added purified water to the antler for 1 to 10 hours at 95 to 105 ℃. The method according to claim 1,
The protease is a protease obtained from a bacterium. The method according to claim 3,
The bacterium is Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis licheniformis ) and Rhizopus sp. The method according to claim 1,
The protease is 0.1 to 5% by weight based on the weight of the antler, characterized in that for 3 to 15 hours the enzyme reaction at 40 to 60 ℃. The method according to claim 5,
The protease is added to 1 to 5% by weight based on the weight of the antler, characterized in that the enzyme reaction at 50 to 55 ℃ for 5 to 15 hours. The method according to claim 1,
The high pressure homogenization is characterized in that for homogenizing 1 to 5 times at 500 to 1,000 bar pressure. The method according to claim 1,
After the high pressure homogenization step, the method further comprising the step of purifying the antler extract through an activated carbon packed column. Antler extract with increased sialic acid content prepared by the method of claim 1. The method according to claim 9,
Sialic acid content of the antler extract is increased sialic acid content, characterized in that 0.15 to 0.35% by weight.

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