Preparation method of fermented pilose antler extract processed through three steps

Abstract

This invention relates to a method for preparing fermented deer antler extract, a fermented deer antler extract prepared by the above method, and a method for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid, and free amino acids in the fermented deer antler extract. The method for preparing fermented deer antler extract is characterized by comprising: step (1) treating pulverized deer antler under high temperature and pressure conditions; step (2) adding a complex enzyme of protease, cellulase, β-glucanase, and hemicellulase to a deer antler liquid containing water treated in step (1) for reaction; step (3) sterilizing the deer antler liquid reacted in step (2), inoculating the sterilized deer antler liquid with Bacillus coagulans culture for fermentation, and then filtering; and step (4) sterilizing the filtrate filtered in step (3), concentrating, and drying it.

Description

Technical Field

[0001] This invention relates to a method for preparing fermented deer antler extract by processing it through three steps: high temperature and pressure treatment, enzyme treatment, and fermentation to increase the content of active ingredients; the fermented deer antler extract prepared by the above method; and a method for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid, and free amino acids in the fermented deer antler extract. Background Technology

[0002] Deer antler (Cervi Parvum Cornu), the unossified young antler of deer, has been a popular tonic in regions centered around Korea, China, and Japan since ancient times, alongside ginseng. The characteristic of this tonic is that its effects are not selectively manifested in specific organs, but rather through a systemic, complex effect. Pharmacological effects of deer antler include promoting protein and nucleic acid synthesis, promoting hematopoiesis, enhancing immunity, providing nutrition to the cardiovascular system, anti-stress, anti-aging, and anti-gastric ulcer effects. Known pharmacological components of deer antler include gangliosides, pantocrin, amino acids, calcium phosphate, calcium carbonate, collagen, phospholipids, chondroitin, glucosamine, and hyaluronic acid.

[0003] Sialic acid, known as a functional and indicative substance in deer antler, is a monosaccharide attached to cells or water-soluble proteins in the form of sugar chains. It is a structural substance that plays an important role in neurotransmitters in the brain, ganglion cell structure, and the structure and function of gangliosides. The representative structural component is N-acetylneuraminic acid.

[0004] Glycosaminoglycans (GAGs) are mucopolysaccharides with a partially sulfonated backbone consisting of repeating uronic acid and glucosamine. In vivo, they exist as proteoglycans. S-GAGs are major components of articular cartilage and the nucleus pulposus of cartilage plates, and are known to have therapeutic effects on arthritis, potent anti-inflammatory effects, and moisture-retaining properties.

[0005] Bacillus coagulans is a Gram-positive facultative anaerobic bacillus that reproduces within the temperature range of 30°C to 55°C, with an optimal reproduction temperature of 50°C. It possesses characteristics of both Bacillus and Lactobacillus genera, exhibiting both sporulation and lactic acid production. Bacillus coagulans is noteworthy as a producer of L-lactic acid, thermostable enzymes, and antimicrobial peptide coagulants because it can produce biomaterials at high temperatures, thus minimizing contamination issues.

[0006] Research on deer antler has been ongoing to this day, with reports focusing on various studies that utilize water-soluble and organic solvents (alcohols, chloroform, ethanol, etc.) for extraction, or utilize proteolytic enzymes and microorganisms to extract the effective components of deer antler, as well as the analysis and exploration of various indicator substances in deer antler.

[0007] Korean Patent No. 1776686 discloses a method for preparing solid preparations of fermented deer antler, and Korean Patent No. 1336292 discloses a method for extracting active ingredients from deer antler using an ultra-high pressure and low temperature extraction method. However, both of these methods differ from the preparation method of the fermented deer antler extract of this invention, which involves three processing steps. Summary of the Invention

[0008] Technical issues

[0009] The present invention is derived from the above requirements. The purpose of the present invention is to provide a method for preparing a fermented deer antler extract containing a large amount of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids by optimizing the preparation conditions such as enzyme treatment and fermentation in order to prepare a fermented deer antler extract with improved effective components.

[0010] Technical solution

[0011] To solve the above problems, the present invention provides a method for preparing fermented deer antler extract, characterized by comprising: step (1), treating pulverized deer antler under high temperature and pressure conditions; step (2), adding a complex enzyme of protease, cellulase, β-glucanase and hemicellulase to the deer antler liquid treated in step (1) with water to react; step (3), sterilizing the deer antler liquid reacted in step (2), inoculating the sterilized deer antler liquid with Bacillus coagulans culture medium for fermentation and then filtering; and step (4), sterilizing the filtrate filtered in step (3), concentrating and drying it.

[0012] Furthermore, the present invention provides a fermented deer antler extract prepared by the above method.

[0013] Furthermore, the present invention provides a method for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids in fermented deer antler extract, comprising: step (1), treating pulverized deer antler under high temperature and pressure conditions; step (2), adding a complex enzyme of protease, cellulase, β-glucanase and hemicellulase to the deer antler liquid in which water is added to the deer antler treated in step (1) for reaction; step (3), sterilizing the deer antler liquid reacted in step (2), inoculating the sterilized deer antler liquid with Bacillus coagulans culture for fermentation and then filtering; and step (4), sterilizing the filtrate filtered in step (3), concentrating and drying it.

[0014] The effects of the invention

[0015] In order to increase the content of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids, which are beneficial components to the human body, this invention prepares fermented deer antler extract through pretreatment, enzyme treatment and fermentation, providing a new form of health-oriented functional food with increased content of effective components. This provides a product different from existing deer antler products and can be effectively used in a variety of health-oriented functional foods. Attached Figure Description

[0016] Figure 1 Photographs showing the degree of solubility of the fermented deer antler extract powder of the present invention after dissolving it in a beaker containing distilled water. Detailed Implementation

[0017] To achieve the objective of this invention, this invention provides a method for preparing fermented deer antler extract, comprising: step (1), treating pulverized deer antler under high temperature and pressure conditions; step (2), adding a complex enzyme of protease, cellulase, β-glucanase and hemicellulase to the deer antler liquid treated in step (1) with added water to react; step (3), sterilizing the deer antler liquid reacted in step (2), inoculating the sterilized deer antler liquid with Bacillus coagulans culture for fermentation and then filtering; and step (4), sterilizing the filtrate filtered in step (3), concentrating and drying it.

[0018] In the preparation method of fermented deer antler extract of the present invention, preferably, step (1) can be performed by treating the pulverized deer antler at a high temperature of 115°C to 125°C and a pressure of 2.5 bar to 3.5 bar for 50 to 70 minutes; more preferably, the pulverized deer antler can be treated at a high temperature of 121°C and a pressure of 3 bar for 60 minutes. Performing high-temperature and high-pressure treatment under the above conditions before enzyme treatment can increase the extraction of effective components to prepare high-quality fermented deer antler extract.

[0019] Furthermore, in the preparation method of the fermented deer antler extract of the present invention, preferably, step (2) above can be performed by adding a complex enzyme consisting of 0.4% to 0.6% (w / v) of protease and 0.4% to 0.6% (w / v) of cellulase, β-glucanase, and hemicellulase relative to the amount of water added to the treated deer antler, and then reacting at a temperature of 55°C to 65°C for 4 to 6 hours. More preferably, it can be performed by adding a complex enzyme consisting of 0.5% (w / v) of protease and 0.5% (w / v) of cellulase, β-glucanase, and hemicellulase relative to the amount of water added to the treated deer antler, and then reacting at a temperature of 65°C for 5 hours. The content of sulfated glycosaminoglycans, N-acetylneuraminic acid, and free amino acids can be greatly increased by using the above-mentioned enzymes for reaction.

[0020] Furthermore, in the preparation method of the fermented deer antler extract of the present invention, preferably, step (3) can be performed by sterilizing the reacting deer antler liquid at a temperature of 110°C to 130°C for 10 to 20 minutes, then inoculating it with 1.5% to 2.5% (v / v) of Bacillus coagulans culture relative to the sterilized deer antler liquid, fermenting it at a temperature of 34°C to 40°C for 20 to 28 hours, and then filtering it. More preferably, it can be performed by sterilizing the reacting deer antler liquid at a temperature of 121°C for 15 minutes, then inoculating it with 2% (v / v) of Bacillus coagulans culture relative to the sterilized deer antler liquid, fermenting it at a temperature of 37°C for 24 hours, and then filtering it. Fermenting enzyme-treated deer antler with the above-mentioned strains can significantly increase the content of effective ingredients while further enhancing the smooth texture and aroma of the deer antler.

[0021] Furthermore, in the preparation method of the fermented deer antler extract of the present invention, preferably, step (4) can be to sterilize the filtrate at a temperature of 90°C to 100°C for 5 to 15 minutes and then concentrate and dry it; more preferably, it can be to sterilize the filtrate at a temperature of 95°C for 10 minutes and then concentrate and dry it.

[0022] More specifically, the preparation method of the fermented deer antler extract of the present invention may include: step (1), treating pulverized deer antler under high temperature of 115°C to 125°C and pressure of 2.5 bar to 3.5 bar for 50 minutes to 70 minutes; step (2), adding 0.4% to 0.6% (w / v) of protease and 0.4% to 0.6% (w / v) of a complex enzyme of cellulase, β-glucanase and hemicellulase relative to the deer antler treated in step (1) to a deer antler extract containing 4 to 6 times (v / w) of water, and then heating for 5 minutes. The reaction is carried out at a temperature of 5℃~65℃ for 4 to 6 hours; in step (3), the deer antler liquid reacted in step (2) is sterilized at a temperature of 110℃~130℃ for 10 to 20 minutes, and then 1.5%~2.5% (v / v) of Bacillus coagulans culture is inoculated relative to the sterilized deer antler liquid and fermented at a temperature of 34℃~40℃ for 20 to 28 hours, and then filtered; and in step (4), the filtrate filtered in step (3) is sterilized at a temperature of 90℃~100℃ for 5 to 15 minutes, then concentrated and dried.

[0023] More specifically, the preparation method of the fermented deer antler extract of the present invention may include: step (1), treating pulverized deer antler under high temperature of 121°C and pressure of 3 bar for 60 minutes; step (2), adding 5 times (v / w) of water to the deer antler liquid treated in step (1), adding 0.5% (w / v) of protease and 0.5% (w / v) of cellulase, β-glucanase and hemicellulase relative to the deer antler liquid, and reacting at 60°C for 5 hours; step (3), sterilizing the deer antler liquid reacted in step (2) at 121°C for 15 minutes, inoculating with 2% (v / v) of Bacillus coagulans culture relative to the sterilized deer antler liquid, fermenting at 37°C for 24 hours, and then filtering; and step (4), sterilizing the filtrate filtered in step (3) at 95°C for 10 minutes, concentrating and drying.

[0024] Furthermore, the present invention provides a fermented deer antler extract prepared by the above method.

[0025] Furthermore, the present invention provides a method for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids in fermented deer antler extract, comprising: step (1), treating pulverized deer antler under high temperature and pressure conditions; step (2), adding a complex enzyme of protease, cellulase, β-glucanase and hemicellulase to the deer antler liquid in which water is added to the deer antler treated in step (1) for reaction; step (3), sterilizing the deer antler liquid reacted in step (2), inoculating the sterilized deer antler liquid with Bacillus coagulans culture for fermentation and then filtering; and step (4), sterilizing the filtrate filtered in step (3), concentrating and drying it.

[0026] More specifically, the method of the present invention for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids in fermented deer antler extract may include: step (1), treating pulverized deer antler under high temperature of 115°C to 125°C and pressure of 2.5 bar to 3.5 bar for 50 to 70 minutes; step (2), adding 0.4% to 0.6% (w / v) of protease and 0.4% to 0.6% (w / v) of cellulase and β-glucanase relative to the deer antler extract to a deer antler extract containing 4 to 6 times (v / v) water treated in step (1) above. After reacting with the complex enzyme of hemicellulase, the reaction is carried out at a temperature of 55℃~65℃ for 4 to 6 hours; in step (3), the deer antler liquid reacted in step (2) is sterilized at a temperature of 110℃~130℃ for 10 to 20 minutes, and then inoculated with 1.5%~2.5% (v / v) of Bacillus coagulans culture relative to the sterilized deer antler liquid and fermented at a temperature of 34℃~40℃ for 20 to 28 hours, and then filtered; and in step (4), the filtrate filtered in step (3) is sterilized at a temperature of 90℃~100℃ for 5 to 15 minutes, then concentrated and dried.

[0027] More specifically, the method of the present invention for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid and free amino acids in fermented deer antler extract may include: step (1), treating pulverized deer antler under high temperature of 121°C and pressure of 3 bar for 60 minutes; step (2), adding 0.5% (w / v) protease and 0.5% (w / v) cellulase relative to the deer antler extract to a deer antler extract containing 5 times (v / w) water of the deer antler treated in step (1). The complex enzymes of β-glucanase and hemicellulase are reacted at 60°C for 5 hours; in step (3), the deer antler liquid reacted in step (2) above is sterilized at 121°C for 15 minutes, and then 2% (v / v) of Bacillus coagulans culture is inoculated relative to the sterilized deer antler liquid and fermented at 37°C for 24 hours before filtration; and in step (4), the filtrate filtered in step (3) above is sterilized at 95°C for 10 minutes, then concentrated and dried.

[0028] The preparation examples and embodiments of the present invention are described in detail below. However, the preparation examples and embodiments described below are only for illustrating the present invention, and the content of the present invention is not limited to the preparation examples and embodiments described below.

[0029] Preparation Example 1. Fermented Deer Antler Extract

[0030] Step (1): Purchase dried deer antler slices, crush them into pieces approximately 0.5cm in length and width using a pulverizer, and process them for 1 hour at a high temperature of 121℃ and a pressure of 3 bar.

[0031] Step (2): Add 5 times (v / w) of pure water to the deer antler liquid treated in step (1) above, add 0.5% (w / v) of protease proteAX as a protease and 0.5% (w / v) of a complex enzyme (a complex enzyme of cellulase, β-glucanase and hemicellulase, Sumizyme brand) relative to the deer antler liquid, and react in a constant temperature water bath at 60°C for 5 hours.

[0032] Step (3): After sterilizing the deer antler liquid reacted in step (2) at 121°C for 15 minutes, inoculate with 2% (v / v) Bacillus coagulans culture medium (10g) relative to the deer antler liquid. 7 cfu / ml-10 8 The mixture was fermented at 37°C for 24 hours in a constant temperature incubator (cfu / ml), and then filtered using filter paper.

[0033] Step (4): Sterilize the filtrate filtered in step (3) at 95°C for 10 minutes, concentrate it using a rotary concentrator, and then freeze-dry it.

[0034] Comparative Example 1. Fermented Deer Antler Extract

[0035] Step (1): Purchase dried deer antler slices and crush them into pieces approximately 0.5 cm in length and width using a grinder. Add 0.5% (w / v) of protease (proteAX) and 0.5% (w / v) of a complex enzyme (a complex enzyme of cellulase, β-glucanase, and hemicellulase, Sumizyme brand) relative to the deer antler liquid containing 5 times (v / w) pure water. Then react in a constant temperature water bath at 60°C for 5 hours.

[0036] Step (2): After sterilizing the deer antler liquid reacted in step (2) at 121°C for 15 minutes, inoculate with 2% (v / v) Bacillus coagulans culture medium (10g) relative to the deer antler liquid. 7 cfu / ml-10 8 The mixture was fermented at 37°C for 24 hours in a constant temperature incubator (cfu / ml), and then filtered using filter paper.

[0037] Step (3): Sterilize the filtrate filtered in step (2) at 95°C for 10 minutes, concentrate it using a rotary concentrator, and then freeze-dry it.

[0038] Comparative Example 2. Fermented Deer Antler Extract

[0039] Step (1): Purchase dried deer antler slices and use a grinder to grind them into pieces approximately 0.5cm in length and width.

[0040] Step (2): Add 5 times (v / w) of pure water to the deer antler extract treated in step (1) above, and add 0.5% (w / v) of protease proteAX (as a proteolytic enzyme) and 0.5% (w / v) of enzyme relative to the deer antler extract. (glucoamylase) was then reacted in a constant temperature water bath at 60°C for 5 hours.

[0041] Step (3): After sterilizing the deer antler liquid reacted in step (2) at 121°C for 15 minutes, inoculate with 2% (v / v) Bacillus coagulans culture medium (10g) relative to the deer antler liquid. 7 cfu / ml-10 8 The mixture was fermented at 37°C for 24 hours in a constant temperature incubator (cfu / ml), and then filtered using filter paper.

[0042] Step (4): Sterilize the filtrate filtered in step (3) at 95°C for 10 minutes, concentrate it using a rotary concentrator, and then freeze-dry it.

[0043] Comparative Example 3. Fermented Deer Antler Extract

[0044] Step (1): Purchase dried deer antler slices, crush them into pieces approximately 0.5cm in length and width using a pulverizer, and process them at a high temperature of 121℃ and a pressure of 3 bar for 1 hour.

[0045] Step (2): Add 1% (w / v) of a mixture of pectinase and cellulase relative to the deer antler liquid to the deer antler liquid treated in step (1) above, and react in a constant temperature water bath at 60°C for 5 hours.

[0046] Step (3): After sterilizing the deer antler liquid reacted in step (2) at 121°C for 15 minutes, inoculate the deer antler liquid with a mixed lactic acid bacteria culture of 2% (v / v) of Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus longum (10 μL). 7 cfu / ml-10 8 The mixture was fermented at 37°C for 24 hours in a constant temperature incubator (cfu / ml), and then filtered using filter paper.

[0047] Step (4): Sterilize the filtrate filtered in step (3) at 95°C for 10 minutes, concentrate it using a rotary concentrator, and then freeze-dry it.

[0048] Comparative Example 4. Deer Antler Extract

[0049] Step (1): Purchase dried deer antler slices, crush them into pieces approximately 0.5cm in length and width using a pulverizer, and process them at a high temperature of 121℃ and a pressure of 3 bar for 1 hour.

[0050] Step (2): Add 1% of a compound enzyme (Novozymes) relative to the deer antler liquid to the deer antler liquid treated in step (1) above with 5 times (v / w) water, and react in a constant temperature water bath at 60°C for 5 hours.

[0051] Step (3): Sterilize the deer antler liquid reacted in step (2) at 95°C for 10 minutes, concentrate it using a rotary concentrator, and then freeze-dry it.

[0052] Experimental Materials and Methods

[0053] 1. Experimental Materials

[0054] The deer antlers were purchased as dried deer antler slices from Russia and pulverized into pieces approximately 0.5 cm in length and width using a grinder. The enzyme preparations used were proteolytic enzyme ProteAX (1400 units / g) and the complex enzyme Sumizyme (200 GAU / g).

[0055] 2. Use of strain isolation and culture medium

[0056] The strain used in fermenting deer antler was Bacillus coagulans (ATCC 7050) obtained from the biological resource center. After culturing Bacillus coagulans in lactic acid bacteria-specific MRS broth at 37°C for 24 hours, it was then stored at 4°C before use.

[0057] 3. Preparation of fermented deer antler extract

[0058] 1) High temperature and pressure treatment method

[0059] The pulverized deer antlers were processed under different pressure conditions and for different processing times.

[0060] 2) Enzyme treatment

[0061] Five times (w / w) of pure water were added to the deer antler that had been treated with high temperature and pressure, and then 0.5% (w / v) of enzyme preparations proteAX and sumizyme were added respectively, and the mixture was reacted in a constant temperature water bath at 60°C for 5 hours.

[0062] 3) Preparation of fermented deer antler extract via fermentation

[0063] After sterilizing the fermented deer antler broth at 121°C for 15 minutes, the solution was cooled to room temperature, inoculated with 2% (v / v) Bacillus coagulans culture, and incubated at 37°C for 24 hours in a thermostat (HB-102L, Korea). The filtrate was then filtered using Whatman no. 3 filter paper (Whatman Co., Maidstone, England). A portion of this filtrate was used as the fermented deer antler extract in experiments. The remaining filtrate was sterilized at 95°C for 10 minutes, concentrated using a rotary concentrator (N-1000VW, Eyela Co., Tokyo, Japan), and then freeze-dried (NB-504, Ilshin Co., Dongducheon, Korea) to be stored as a powder at room temperature.

[0064] 4. Analyze the project

[0065] 1) pH, sugar content, and color

[0066] pH was measured using a pH meter (Orion Star A111, Thermo Scientific, USA), and saccharimetric content was measured using a refractometer saccharimeter (PR-101, Atago Co. Ltd., Japan). Colorimetry was measured using a UV-visible spectrometer (UV-1601, Shimadzu, Japan).

[0067] 2) Soluble solids content

[0068] After taking 1g of sample, the sample was evaporated and dried at 105°C using a moisture analyzer (FD-660, Kett Corporation, South Korea). The weight of the sample was then measured, and the soluble solids content was expressed as the dry matter weight relative to the sample weight.

[0069] 3) Free amino acid content

[0070] An equal volume of 5% trichloroacetic acid (TCA) solution was added to 10 g of sample and stirred for 1 hour. The mixture was then centrifuged at 10,000 rpm for 10 minutes to remove proteins, and the supernatant was collected. The supernatant was then extracted three times with 100 mL of diethyl ether to remove lipids, pigments, and lipid-soluble substances. The aqueous layer was concentrated under reduced pressure at 40°C and dissolved in 10 mL of 0.2 N lithium citrate buffer (pH 2.2). The solution was then filtered through a membrane filter (0.2 μm pore size, Advantec MFS, Japan) and analyzed using an automated amino acid analyzer (Biochem 20, Pharmacia Biotech Ltd., England). Results are expressed as mg% relative to the extract.

[0071] 4) Content of sulfonated glycosaminoglycans (GAGs)

[0072] The determination of sulfated glycosaminoglycans began by dissolving 1.185 g of NaCl, 1.52 g of glycine, 0.008 g of dimethylmethylene blue (DMB), and 0.47 mL of 36% hydrochloric acid (HCl) in 500 mL of distilled water to prepare a DMB dye solution. The prepared solution exhibited an absorbance of approximately 0.34 at 525 nm. 0.2 mL of the sample was mixed with 8 mL of the DMB solution, and the absorbance was measured at 525 nm. The content of sulfated glycosaminoglycans in the sample was calculated using a calibration curve based on chondroitin 4-sulfate and expressed as mg% relative to the extract.

[0073] 5) N-acetylneuraminic acid content

[0074] The N-acetylneuraminic acid content was measured by adding 15 mM hydrochloric acid solution to the sample and then hydrolyzing it in a constant temperature water bath at 80°C for 3 hours to release sialic acid. After the reaction, the filtrate filtered through a 0.45 μm syringe filter was analyzed by high performance liquid chromatography (HPLC).

[0075] 6) Water solubilization of fermented deer antler extract powder

[0076] After weighing 0.5g of fermented deer antler extract powder prepared by high temperature and pressure pretreatment, the dissolution time in a 100mL beaker containing 80mL distilled water was measured to confirm water solubility.

[0077] 7) Storage stability of fermented deer antler extract powder

[0078] To confirm the storage stability of the fermented deer antler extract powder prepared after high-temperature and pressure pretreatment, an accelerated test was conducted. After storage at 50°C for 6 months, the number of microorganisms and pH were measured. The number of microorganisms was measured by counting common bacteria and Escherichia coli, while the pH was measured using a pH meter after adding a specified amount of water to the fermented deer antler extract powder.

[0079] Example 1. Characteristics of fermented deer antler extract under high temperature and pressure treatment conditions

[0080] The results of comparing the physicochemical properties of the fermented deer antler extract with those of the fermented deer antler extract prepared in Example 1 under the high temperature and pressure treatment conditions of step (1) are shown in Table 1 below.

[0081] Table 1

[0082] Physicochemical properties of fermented deer antler extract under different high temperature and pressure conditions

[0083]

[0084]

[0085] The pH value tends to decrease with increasing pressure and processing time. This result suggests that under high temperature and pressure conditions, as the physical properties of the raw materials change, the extracted active ingredients contain more acidic components. Sugar content and soluble solids content also increase with increasing pressure and processing time. In colorimetry, L represents brightness, a represents redness, and b represents yellowness; redness increases with increasing pressure and processing time.

[0086] Example 2. Content of effective components in fermented deer antler extract under high temperature and pressure treatment conditions

[0087] The fermented deer antler extract of Preparation Example 1 and the fermented deer antler extract prepared by the method of Preparation Example 1 were compared. The results of comparing the contents of sulfated glycosaminoglycans (s-GAG) and N-acetylneuraminic acid under different high temperature and pressure treatment conditions in step (1) are shown in Table 2 below.

[0088] s-GAG refers to glycosaminoglycans with added sulfonic acid groups. Glycosaminoglycans are lactoses formed by repeating structures of amino sugars with uronic acid or galactose, and are known to be essential components of animal tissues and connective tissues. Sulfated glycosaminoglycans are a major component of articular cartilage and have therapeutic effects on arthritis by maintaining intra-articular moisture. They also link with proteins to form proteoglycans, which are found in various connective tissues such as skin and umbilical cord, playing a role in maintaining the health of these tissues through structural formation and functional regulation.

[0089] N-acetylneuraminic acid is a monosaccharide that is also found in glycoproteins in cells, cell membranes, and mucus, as well as in glycolipids such as gangliosides, which are important structural components of nerve cell membranes in the brain. It is a representative substance of sialic acid.

[0090] Table 2

[0091] The content of active ingredients in fermented deer antler extract varies depending on different high temperature and pressure conditions.

[0092]

[0093]

[0094] The results showed that the highest content of active ingredients was obtained by treating the product at 121°C and 3 bar for 1 hour.

[0095] Example 3. Content of active ingredients in fermented deer antler extract under varying processing conditions

[0096] The results of comparing the content of effective components in fermented deer antler extract based on whether high temperature and pressure treatment were used, the type of enzyme, and the type of strain are shown in Table 4 below.

[0097] Table 3

[0098] Comparison of processing conditions for fermented deer antler extract

[0099]

[0100] The results showed that, compared with the fermented deer antler extract of the comparative example, the fermented deer antler extract of Preparation Example 1 showed the highest content of s-GAG and N-acetylneuraminic acid, while the fermented deer antler extract of Comparative Example 1 showed the lowest content.

[0101] Table 4

[0102] The content of active ingredients in fermented deer antler extract varies depending on processing conditions.

[0103] distinguish s-GAG content (mg%) N-acetylneuraminic acid content (ppm) Preparation Example 1 34.13 100.04 Comparative Example 1 16.87 38.99 Comparative Example 2 13.20 35.40 Comparative Example 3 20.15 44.78 Comparative Example 4 18.00 39.20

[0104] Example 4. Free amino acid content of fermented deer antler extract under varying processing conditions

[0105] The results of comparing the free amino acid content of fermented deer antler extract under different processing conditions are shown in Table 5 below.

[0106] Table 5

[0107] The content of active ingredients in fermented deer antler extract varies depending on processing conditions.

[0108]

[0109]

[0110]

[0111] The results showed that the fermented deer antler extract of Comparative Example 2 had the lowest content, while the fermented deer antler extract of Preparation Example 1 had the highest content. The sum of the eight essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, and lysine) was also highest in the fermented deer antler extract of Preparation Example 1.

[0112] Essential amino acids cannot be synthesized in the body and must be obtained through diet or supplements. Leucine, in particular, promotes protein synthesis, contributing to muscle growth and skeletal muscle strengthening. Lysine is involved in calcium metabolism, aiding bone development by promoting calcium absorption and maintaining nitrogen balance. When ingested with vitamin C, it synthesizes collagen, a necessary component for maintaining skin and blood vessel elasticity and for cartilage formation. Phenylalanine is essential for the production of neurotransmitters such as tyrosine, dopamine, epinephrine, and norepinephrine, and is known to contribute to brain cell regeneration and protein production. The fermented deer antler extract in Preparation Example 1 was found to contain high levels of these amino acids, suggesting they may contribute to physical growth and brain development.

[0113] Example 4. Water solubilization of fermented deer antler extract

[0114] Regarding the water solubility of the fermented deer antler extract in Preparation Example 1, as follows: Figure 1 As shown in the figure. Immediately after adding the fermented deer antler extract powder pretreated under high temperature and pressure, the water solubility was checked at 30-second intervals until 1 minute and 30 seconds later. It was confirmed that a small amount of powder began to dissolve immediately after addition, and after 30 seconds, half of the powder had dissolved. Then, after about 1 minute, most of it had dissolved, and in the final 1 minute and 30 seconds, it was completely dissolved, as the lipid components of deer antler exhibited a layer separation phenomenon. Therefore, it is determined that the fermented deer antler extract powder pretreated under high temperature and pressure, due to its rapid water solubility, can be used as a material and in various dosage forms in a variety of products. The low molecular weight of the active ingredients due to pressure treatment facilitates absorption in the body.

[0115] Example 5. Storage stability of fermented deer antler extract

[0116] The results confirming the storage stability of the fermented deer antler extract powder prepared in Preparation Example 1 are shown in Table 6. The number of common bacteria was negative or 1 CFU / g during different storage periods, and the number of Escherichia coli was negative. The pH during storage ranged from 5.71 to 5.78, indicating that the powder product was stable during storage.

[0117] Table 6

[0118] Storage stability of fermented deer antler extract

[0119]

[0120] *: Not detected.

Claims

1. A method for preparing fermented deer antler extract, characterized in that, include: Step (1): Process the pulverized deer antler at a high temperature of 115℃~125℃ and a pressure of 2.5 bar~3.5 bar for 50 minutes~70 minutes; Step (2): Add 0.4% to 0.6% w / v of protease and 0.4% to 0.6% w / v of a complex enzyme composed of cellulase, β-glucanase and hemicellulase relative to the amount of water added to the deer antler treated in step (1) above, and react at a temperature of 55°C to 65°C for 4 to 6 hours. Step (3): After sterilizing the deer antler liquid reacted in step (2) at a temperature of 110℃~130℃ for 10 to 20 minutes, inoculate the sterilized deer antler liquid with 1.5%~2.5% v / v Bacillus coagulans culture and ferment at a temperature of 34℃~40℃ for 20 to 28 hours, then filter; and Step (4): The filtrate filtered in step (3) above is sterilized, concentrated, and dried.

2. The method for preparing fermented deer antler extract according to claim 1, characterized in that, Step (4) involves sterilizing the filtrate filtered in step (3) at a temperature of 90℃ to 100℃ for 5 to 15 minutes, then concentrating and drying it.

3. A fermented deer antler extract, characterized in that, Prepared by the method according to claim 1 or 2.

4. A method for increasing the content of sulfated glycosaminoglycans, N-acetylneuraminic acid, and free amino acids in fermented deer antler extract, comprising: Step (1): Process the pulverized deer antler at a high temperature of 115℃~125℃ and a pressure of 2.5 bar~3.5 bar for 50 minutes~70 minutes; Step (2): Add 0.4% to 0.6% w / v of protease and 0.4% to 0.6% w / v of a complex enzyme composed of cellulase, β-glucanase and hemicellulase relative to the amount of water added to the deer antler treated in step (1) above, and react at a temperature of 55°C to 65°C for 4 to 6 hours. Step (3): After sterilizing the deer antler liquid reacted in step (2) at a temperature of 110℃~130℃ for 10 to 20 minutes, inoculate the sterilized deer antler liquid with 1.5%~2.5% v / v Bacillus coagulans culture and ferment at a temperature of 34℃~40℃ for 20 to 28 hours, then filter; and Step (4): Sterilize the filtrate filtered in step (3) at a temperature of 90℃~100℃ for 5 to 15 minutes, then concentrate and dry it.

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