A pilose antler element, its preparation method and application

Abstract

The application discloses a kind of pilose antler element and its preparation method and application, it is related to pilose antler element preparation technical field.The application provides a kind of preparation method of pilose antler element, comprising the following steps: (1) the unossified fresh pilose antler section seal is washed after being closed, dry after first freezing, after slicing, immerse in anhydrous ethanol, dry after second freezing, obtain pilose antler section;(2) the pilose antler section is continued to freeze-drying after being crushed, obtain pilose antler micro powder, after pilose antler micro powder is dissolved in water, freeze, after recovery to 15-25 DEG C, obtain mixed solution;(3) lactic acid bacteria and yeast are added to the mixed solution and fermented, after fermentation is broken down, obtain pilose antler slurry;Wherein, fermentation is 2-stage fermentation, first by anaerobic fermentation of lactic acid bacteria, then by aerobic fermentation of yeast;(4) the pilose antler slurry is freeze-dried, and the pilose antler element is obtained.

Description

Technical Field

[0001] This invention relates to the field of deer antler preparation technology, and in particular to a deer antler preparation method and its application. Background Technology

[0002] Deer antlers are the unossified, densely haired young antlers of male sika deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus). They contain pre-osteoblastic tissue such as collagen and are rich in blood vessels and nerves. Ancient physicians believed that the essence of a deer resides entirely in its antlers, and antlers, being the tender shoots of the antlers, possess complete and unreleased energy, thus having the strongest effect in tonifying yang and nourishing blood. Li Shizhen of the Ming Dynasty stated in his *Compendium of Materia Medica* that deer antlers "are excellent at tonifying the kidneys and strengthening yang, generating essence and nourishing blood, and replenishing marrow and strengthening bones." Modern scientific research has proven that deer antler contains more than 20 kinds of amino acids, various hormones, superoxide dismutase (SOD), and polyamines. It also contains phospholipids, polysaccharides, polypeptides, vitamin A, deer antler insulin-like growth factor (IGF-1), deer antler growth hormone (HGH), deer antler growth-releasing factor (GHRF), deer antler nerve growth factor (NGF), deer antler epidermal growth factor (EGF), deer antler fibroblast growth factor (FGF), and many other bioactive peptides, as well as calcium phosphate, chondroitin sulfate, and many other bioactive factors. Deer antler is a sweet, salty, and warm-natured medicine that enters the liver and kidney meridians. It has the effects of tonifying the kidneys and strengthening yang, benefiting essence and marrow, strengthening tendons and bones, replenishing qi and blood, and unblocking the Du meridian. It can promote human growth and development and metabolism, improve the body's antioxidant capacity, and delay aging; alleviate sexual dysfunction in adolescence and prostate atrophy in middle-aged and elderly people, and improve sexual function; enhance the regeneration process of ulcers that are slow to heal or poorly formed, and promote fracture healing; it also has various effects such as anti-stress, anti-inflammatory, and anti-tumor properties. Studies have also confirmed that deer antler can enhance the body's cellular and humoral immune functions, promote lymphocyte transformation, and act as an immune booster, making it an excellent raw material for traditional Chinese medicine and health products.

[0003] The main purpose of deer antler processing is to dehydrate, dry, preserve, disinfect, maintain shape and color, improve quality, and facilitate preservation. For a long time, my country's deer antler processing has relied on boiling and high-temperature baking techniques. However, prolonged high-temperature boiling and frying can lead to the loss of many water-soluble components, as well as the varying degrees of loss, damage, or complete destruction of heat-sensitive active ingredients such as proteins and polypeptides, resulting in a decline in product quality. Defects such as broken skin, hollow parts, rancidity, charring, and spoilage often occur, affecting medicinal efficacy and causing economic losses.

[0004] The processing method for deer antler slices recorded in Part I of the 2010 edition of the Pharmacopoeia of the People's Republic of China is to soak the deer antler in hot white wine or steam it briefly in white wine before slicing it. The disadvantages are that the difficulty of slicing increases with the degree of ossification of the deer antler, the porosity of the slices is uneven, and the slices are thick and brittle. When decocting the medicine, the water-soluble proteins, total phospholipids and total polysaccharides of the deer antler slices are not easy to dissolve. When taken orally, the deer antler slice residue is coarse and has a poor taste.

[0005] Deer antler lozenges are favored by consumers as health products. However, because deer antler contains rich protein, polypeptides, and amino acids, these substances are also prone to complex reactions under the action of microbial decomposition enzymes, forming a variety of substances with unpleasant odors, which causes the deer antler extract to emit a fishy and foul smell. Some literature and patents describe water or alcohol extraction of deer antler powder. For example, patent application CN110063967A, "Preparation Method of Deer Antler Extract, Deer Antler Extract and Its Application," mentions using a solvent reflux method to extract the effective components from deer antler at an extraction temperature of 80-85℃. Patent application CN112715935A, "A Deer Antler Lozenge and Its Preparation Method," mentions using ultrasonic water extraction + alcohol extraction to extract deer antler, followed by compounding with microcrystalline cellulose, sorbitol, lactose, croscarmellose sodium, magnesium stearate, etc. Patent application CN108969538A, "A Production Process of Deer Antler Lozenges and Their Derivative Deer Antler Polypeptides," uses a two-stage water bath extraction of deer antler active ingredients at an extraction temperature not lower than 80℃. Patent authorization announcement CN106551391B, "A Deep Processing Method for Deer Antler," uses protease to enzymatically hydrolyze deer antler to obtain deer antler bone powder, and then prepares deossified deer antler liposomes. The above methods have solved the problem of the fishy and foul odor of deer antler, but they all have the drawbacks of complex processes and loss of a lot of active ingredients. The main reason is that the high temperature, organic solvents, enzymes and other factors used in the extraction process can destroy the effective components of deer antler. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a deer antler extract, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing deer antler extract, comprising the following steps:

[0008] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, slice it, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0009] (2) The deer antler slices are freeze-dried and then pulverized to obtain deer antler powder. The deer antler powder is dissolved in water and then frozen, and then restored to 15-25℃ to obtain a mixture.

[0010] (3) Add lactic acid bacteria and yeast to the mixture for fermentation, and crush it after fermentation to obtain deer antler slurry; wherein, the fermentation is a two-stage fermentation, first by anaerobic fermentation by lactic acid bacteria, and then by aerobic fermentation by yeast;

[0011] (4) After freeze-drying the deer antler slurry, the deer antler extract is obtained.

[0012] Preferably, in step (1), the fresh deer antlers are selected from healthy 3-6 year old male sika deer, and the antler harvesting time is two weeks before and after the Dragon Boat Festival each year.

[0013] Preferably, in step (1), the sealing method is one of starch sealing, chitosan powder sealing, flame baking sealing, and electric soldering iron sealing; more preferably, in step (1), the sealing method is food-grade chitosan powder sealing, with chitosan powder evenly sprinkled on the cut surface until no visible fresh blood is visible.

[0014] The purpose of this invention's sealing mechanism is to preserve the velvet blood, which is rich in amino acids, phospholipids, minerals, collagen, proteoglycans, chondroitin sulfate, glucosamine sulfate, glucosamine polysaccharide, hyaluronic acid, nucleotides, gangliosides, growth hormones, and auxins, and its loss should be prevented. This invention preferably uses food-grade chitosan powder for sealing because food-grade chitosan is derived from chitin and is the only naturally occurring polysaccharide with a positive charge, possessing certain antibacterial and preservative properties.

[0015] Preferably, in step (1), the cleaning is performed using an ultrasonic cleaner with a frequency of 20-40KHz, a power of 100-200W, a cleaning time of 5-15min, and a cleaning temperature of 15-30℃; the drying time is 1-3h, and the drying temperature is 20-25℃; the first freezing is performed using liquid nitrogen, with a freezing time of 30-60min, and the immersion time in anhydrous ethanol is 15-30min; the second freezing is performed using liquid nitrogen, with a freezing time of 1-3h.

[0016] The purpose of this invention is to clean the surface of the deer antlers, removing dirt, impurities, and parasitic microorganisms. Soaking in anhydrous ethanol loosens the structure of the antler slices.

[0017] More preferably, the thickness of the sliced ​​deer antler is 2-5 mm, and the liquid nitrogen freezing is immersion freezing.

[0018] Preferably, in step (2), the antler powder is freeze-dried to a moisture content of ≤15%, and pulverized to a particle size greater than 400 mesh >70%. The mass ratio of the antler powder to water is antler powder:water = 1:(5-10). The water is preferably ultrapure water with a resistivity of 18 MΩ*cm (25℃). In step (2), the freezing time after the antler powder is dissolved in water is 48-72h, and the freezing temperature is -10~-30℃.

[0019] The purpose of freezing the deer antler powder after dissolving it in water is to increase the volume of water contained in the deer antler cells at low temperatures, making the cell structure loose and facilitating subsequent fermentation and cell disruption.

[0020] Preferably, in step (2), the freeze-drying equipment is a freeze dryer with a working pressure of 0.4-1.0 MPa, and the freeze-drying time is 3-8 hours.

[0021] Preferably, in step (3), the lactic acid bacteria include Bifidobacterium and Lactobacillus, and the inoculation amount of Bifidobacterium is 3 × 10⁻⁶. 5 -10×10 7 CFU / ml, the inoculum size of the lactobacillus is 1.5 × 10⁻⁶. 5 -5.0×10 7 CFU / ml; the yeast strain is at least one of *Candida utilis*, *Pichia pastoris*, *Kluyveromyces kusnezoffii*, and *Cryptospira spp.*, and the inoculum size is 1.0 × 10⁻⁶. 5 -1.0×10 7 CFU / ml;

[0022] More preferably, the Bifidobacterium is Bifidobacterium longum. (Bifidobacterium longum) Bifidobacterium longum subsp. (Bifidobacterium longum subsp. longum Reuter), Bifidobacterium infantis (Bifidobacterium infantis) Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium adolescentis (Bifidobacterium adolensentis) Bifidobacterium bifidum (Bifidobacterium bifidum) At least one of the following; the lactobacillus is Lactobacillus acidophilus. (Lactobacillus acidophilus) Lactobacillus paracasei (Lactobacillus paracasei) Lactobacillus rhamnosus (Lactobacillus rhamnosus) , Lactobacillus brevis (Lactobacillus brevis) Lactobacillus bulgaricus (Lactobacillus bulgaricus) Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus helveticus (lactobacillus helveticus) Lactobacillus plantarum (Lactobacillus plantarum) At least one of them.

[0023] Preferably, in step (3), during the fermentation process, those skilled in the art can add, as needed, the culture medium substances required by the fermentation bacteria, such as carbon sources and nitrogen sources, to the deer antler powder solution. The carbon source is at least one of glucose, lactose, galactose, trehalose, dextrin, maltose, maltitol, and mannose; the nitrogen source is at least one of milk protein, silk fibroin, wheat protein, oat protein, and corn protein.

[0024] Preferably, in step (3), lactose is added to the mixture as a carbon source, and the amount of lactose added is 0.1-0.2% of the mass of the mixture. Milk protein is added to the deer antler powder solution as a nitrogen source, and the amount of milk protein added is 0.15-0.25% of the mass of the deer antler powder solution.

[0025] Those skilled in the art can adjust specific conditions such as fermentation temperature, time, stirring speed, and oxygen supply according to the type and amount of fermenting bacteria used.

[0026] Preferably, in step (3), when lactic acid bacteria undergo anaerobic fermentation, the fermentation temperature is 20-45℃, the fermentation time is 3-8h, and the stirring speed is 50-100rpm; when yeast undergoes aerobic fermentation, the fermentation temperature is 30-42℃, the fermentation time is 2-6h, the stirring speed is 10-50rpm, and the oxygen content of the fermentation liquid is 0.5-1.0mg / L.

[0027] More preferably, during anaerobic fermentation of lactic acid bacteria, the fermentation temperature is 30-40℃, the fermentation time is 4-6h, and the stirring speed is 60-80rpm; during aerobic fermentation of yeast, the fermentation temperature is 35-40℃, the fermentation time is 3-5h, and the stirring speed is 20-40rpm.

[0028] The compound fermentation of this invention facilitates the hydrolysis of high molecular weight deer antler proteins, polysaccharides and other substances, generating small molecule peptides, polysaccharides and other degradation products, greatly increasing the content of polypeptides with a molecular weight ≤1000 Daltons in the final deer antler extract, while also obtaining active substances such as amino acids, biological enzymes, and nucleotides.

[0029] Preferably, in step (3), the crushing process uses a low-temperature ultra-high pressure continuous flow cell disruptor. Ultra-high pressure energy is used to release the sample instantaneously through a slit, causing cell breakage, homogenization, and dispersion under the influence of shearing, cavitation, and collision effects. The material temperature is 4-6℃ and the pressure is 207MPa during operation; the low-temperature ultra-high pressure continuous flow cell disruptor is used for 2-5 cycles.

[0030] Preferably, in step (4), the freeze-drying time is 6-8 hours, the moisture content of the deer antler extract is ≤1%, and the component with a particle size of less than 100 μm is >99%.

[0031] Furthermore, the present invention provides deer antler extract prepared using the above-described method for preparing deer antler extract.

[0032] Furthermore, the present invention provides the application of the aforementioned deer antler extract in the preparation of pharmaceuticals.

[0033] Preferably, when the deer antler extract is used, excipients such as dextrin, starch, microcrystalline cellulose, mannan, etc. may be added. The dosage form of the deer antler extract of the present invention is one of tablets, powders, granules, and capsules.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) The deer antler extract provided by this invention contains all the nutritional components of deer antler, including protein, polypeptides, amino acids, polysaccharides, fatty acids, steroids, phospholipids, nucleosides, biogenic amines, and trace elements. Specifically, the deer antler extract provided by this invention contains all the nutritional components of deer antler, including 64.72-68.55% protein, 2.2-3.4% total water-soluble amino acids, 2600-3000 mg / Kg total water-soluble polysaccharides, 1.1-1.8% fat, 4.0-6.5 g / Kg total nucleosides, 18-32% ash, and moisture ≤1%. (2) The entire processing of this invention is carried out at room temperature or below room temperature, with the temperature not exceeding 30°C, thus avoiding the destruction of heat-sensitive active ingredients in deer antler. (3) The processing technology does not involve water extraction, solvent extraction, baking, or other methods, thus preserving all the active ingredients in deer antler without loss. (4) Based on conventional ultrafine grinding, this invention combines symbiotic fermentation of compound microorganisms and low-temperature ultra-high pressure continuous flow cell disruption technology, resulting in a high content of active ingredients in the deer antler powder. (5) The deer antler powder obtained by this invention has a high degree of protein hydrolysis, good taste and absorption, and is convenient to use. Attached Figure Description

[0036] Figure 1 The diagrams are tissue morphology diagrams; where (a) is the tissue morphology diagram of the blank group, (b) is the tissue morphology diagram of Example 1, and (c) is the tissue morphology diagram of Comparative Example 8.

[0037] Figure 2 The images are FLG immunofluorescence patterns; (d) is the FLG immunofluorescence pattern of the blank group, (e) is the FLG immunofluorescence pattern of Example 1, and (f) is the FLG immunofluorescence pattern of Comparative Example 8.

[0038] Figure 3 The images are LOR immunofluorescence patterns; (g) is the LOR immunofluorescence pattern of the blank group, (h) is the LOR immunofluorescence pattern of Example 1, and (i) is the LOR immunofluorescence pattern of Comparative Example 8. Detailed Implementation

[0039] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0040] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0041] The lactic acid bacteria and yeast strains used in this invention are both standard strains, purchased from the Guangdong Institute of Microbiology.

[0042] Example 1

[0043] A method for preparing deer antler extract includes the following steps:

[0044] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 2-5 mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0045] The fresh deer antlers were selected from healthy 4-year-old male sika deer, and the antlers were sealed with chitosan powder. The cleaning process involved an ultrasonic cleaner with a frequency of 40kHz and a power of 100W, a cleaning time of 10 minutes, and a cleaning temperature of 20℃. The drying time was 2 hours, and the drying temperature was 20℃. The first freezing was done with liquid nitrogen for 45 minutes, followed by immersion in anhydrous ethanol for 20 minutes. The second freezing was also done with liquid nitrogen for 2 hours.

[0046] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 20°C to obtain a mixture. The freeze-drying is carried out until the moisture content is 8%. The freeze-drying equipment is a freeze dryer with a working pressure of 0.6 MPa and the freeze-drying time is 4 hours. The pulverized powder is pulverized to 80% of the components with a particle size greater than 400 mesh. The mass ratio of deer antler powder to water is deer antler powder: water = 1:8. In step (2), the time for freezing the deer antler powder after dissolving it in water is 60 hours and the freezing temperature is -20°C.

[0047] (3) Lactic acid bacteria and yeast are added to the mixture for fermentation. After fermentation, the mixture is crushed three times using a low-temperature, ultra-high-pressure continuous flow cell crusher to obtain deer antler slurry. The fermentation is a two-stage process: first, anaerobic fermentation is carried out by lactic acid bacteria, and then aerobic fermentation is carried out by yeast. The lactic acid bacteria include bifidobacteria and lactobacilli, and the inoculation amount of bifidobacteria is 8 × 10⁻⁶. 6 CFU / ml, the inoculum size of the lactobacillus is 4 × 10⁻⁶. 6CFU / ml; the yeast strain is *Diplostomum cuspidatum*, and the inoculum size is 1.0 × 10⁻⁶. 6 CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.15% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.2% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 35℃, the fermentation time was 3 hours, and the stirring speed was 70 rpm. For aerobic fermentation with yeast, the fermentation temperature was 40℃, the fermentation time was 4 hours, the stirring speed was 15 rpm, and the oxygen content of the fermentation broth was 0.8 mg / L.

[0048] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 0.6 MPa for 7 hours, the deer antler extract has a moisture content of 0.6% and a particle size of less than 100 μm of 99.5%, thus obtaining the deer antler extract.

[0049] Example 2

[0050] A method for preparing deer antler extract includes the following steps:

[0051] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 2mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0052] The fresh deer antlers were selected from healthy 3-year-old male sika deer, and the antlers were sealed with starch. The cleaning process involved an ultrasonic cleaner with a frequency of 30kHz and a power of 200W, lasting 5 minutes at a temperature of 22℃. The drying process took 1 hour at 22℃. The first freezing was done with liquid nitrogen for 30 minutes, followed by immersion in anhydrous ethanol for 15 minutes. The second freezing was also done with liquid nitrogen for 1 hour.

[0053] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 15°C to obtain a mixture. The freeze-drying is carried out until the moisture content is 10%. The freeze-drying equipment is a freeze dryer with a working pressure of 0.4 MPa and the freeze-drying time is 3 hours. The pulverized powder is pulverized to 85% of the components with a particle size greater than 400 mesh. The mass ratio of deer antler powder to water is deer antler powder: water = 1:5. In step (2), the time for freezing the deer antler powder after dissolving it in water is 48 hours and the freezing temperature is -10°C.

[0054] (3) Lactic acid bacteria and yeast are added to the mixture for fermentation. After fermentation, the mixture is crushed twice using a low-temperature ultra-high pressure continuous flow cell crusher to obtain deer antler slurry. The fermentation is a two-stage process, with anaerobic fermentation by lactic acid bacteria followed by aerobic fermentation by yeast. The lactic acid bacteria include Bifidobacterium and Lactobacillus, and the inoculation amount of Bifidobacterium is 3 × 10⁻⁶. 5 CFU / ml, the inoculum size of the lactobacillus is 1.5 × 10⁻⁶. 5 CFU / ml; the yeast strain is *Candida utilis*, and the inoculum size is 1.0 × 10⁻⁶. 5 CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.1% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.15% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 20℃, the fermentation time was 4 hours, and the stirring speed was 50 rpm. For aerobic fermentation with yeast, the fermentation temperature was 30℃, the fermentation time was 2 hours, the stirring speed was 10 rpm, and the oxygen content of the fermentation broth was 0.5 mg / L.

[0055] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 0.4 MPa for 6 hours, the deer antler extract has a moisture content of 0.7% and a particle size of less than 100 μm of 99.3%, and the deer antler extract is obtained.

[0056] Example 3

[0057] A method for preparing deer antler extract includes the following steps:

[0058] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 5mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0059] The fresh deer antlers were selected from healthy 5-year-old male sika deer, and the antlers were sealed with starch. The cleaning process involved an ultrasonic cleaner with a frequency of 20kHz and a power of 150W, a cleaning time of 15 minutes, and a cleaning temperature of 25℃. The drying time was 3 hours, and the drying temperature was 25℃. The first freezing was done with liquid nitrogen for 60 minutes, followed by immersion in anhydrous ethanol for 18 minutes. The second freezing was also done with liquid nitrogen for 3 hours.

[0060] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 18°C ​​to obtain a mixture. The freeze-drying is carried out until the moisture content is 9%. The freeze-drying equipment is a freeze dryer with a working pressure of 1.0 MPa and the freeze-drying time is 8 hours. The pulverized powder is pulverized to a particle size greater than 400 mesh, and the mass ratio of deer antler powder to water is deer antler powder: water = 1:10. In step (2), the time for freezing the deer antler powder after dissolving it in water is 50 hours and the freezing temperature is -15°C.

[0061] (3) Add lactic acid bacteria and yeast to the mixture for fermentation. After fermentation, use a low-temperature ultra-high pressure continuous flow cell disruptor to crush the mixture 2-5 times to obtain deer antler slurry. The fermentation is a two-stage process: first, anaerobic fermentation is carried out by lactic acid bacteria, and then aerobic fermentation is carried out by yeast. The lactic acid bacteria include bifidobacteria and lactobacilli, and the inoculation amount of bifidobacteria is 5×10⁻⁶. 6 The inoculum density of the lactobacillus was 2.5 × 10⁻⁶ CFU / ml. 6 CFU / ml; the yeast includes *Candida utilis* and *Candida spp.*, and the inoculum size of *Candida utilis* is 0.5 × 10⁻⁶. 7 The inoculum size of *CFU / ml* was 0.5 × 10⁻⁶ CFU / ml. 7 CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.12% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.18% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 32℃, the fermentation time was 4.5 h, and the stirring speed was 65 rpm. For aerobic fermentation with yeast, the fermentation temperature was 40℃, the fermentation time was 5 h, the stirring speed was 30 rpm, and the oxygen content of the fermentation broth was 0.6 mg / L.

[0062] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 1 MPa for 8 hours, the deer antler extract has a moisture content of 0.8% and a particle size of less than 100 μm of 99.8%, and the deer antler extract is obtained.

[0063] Example 4

[0064] A method for preparing deer antler extract includes the following steps:

[0065] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 4mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0066] The fresh deer antlers were selected from healthy 6-year-old male sika deer, and the antlers were sealed with starch. The cleaning process involved an ultrasonic cleaner with a frequency of 40kHz and a power of 100W, lasting 8 minutes at a temperature of 23℃. The drying process took 1.5 hours at 23℃. The first freezing was done with liquid nitrogen for 40 minutes, followed by immersion in anhydrous ethanol for 25 minutes. The second freezing was also done with liquid nitrogen for 1.5 hours.

[0067] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 20°C to obtain a mixture. The freeze-drying is carried out until the moisture content is 7%. The freeze-drying equipment is a freeze dryer with a working pressure of 0.5 MPa and the freeze-drying time is 5 hours. The pulverized powder is pulverized to 80% of the components with a particle size greater than 400 mesh. The mass ratio of deer antler powder to water is deer antler powder: water = 1:6. In step (2), the time for freezing the deer antler powder after dissolving it in water is 55 hours and the freezing temperature is -25°C.

[0068] (3) Lactic acid bacteria and yeast are added to the mixture for fermentation. After fermentation, the mixture is crushed four times using a low-temperature, ultra-high-pressure continuous flow cell disruptor to obtain deer antler slurry. The fermentation is a two-stage process: first, anaerobic fermentation is carried out by lactic acid bacteria, and then aerobic fermentation is carried out by yeast. The lactic acid bacteria include Bifidobacterium and Lactobacillus, and the inoculation amount of Bifidobacterium is 10 × 10⁻⁶. 7 The inoculum size of the lactobacillus was 5.0 × 10⁻⁶ CFU / ml. 7 CFU / ml; the yeast strain is *Diplostomum cuspidatum*, and the inoculum size is 1.0 × 10⁻⁶. 7 CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.2% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.25% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 45℃, the fermentation time was 8 hours, and the stirring speed was 100 rpm. For aerobic fermentation with yeast, the fermentation temperature was 42℃, the fermentation time was 6 hours, the stirring speed was 20 rpm, and the oxygen content of the fermentation broth was 1.0 mg / L.

[0069] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 0.5 MPa for 6.5 h, the deer antler extract is obtained with a moisture content of 0.5% and a particle size of less than 100 μm of 99.6%.

[0070] Example 5

[0071] A method for preparing deer antler extract includes the following steps:

[0072] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 2.5 mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0073] The fresh deer antlers were selected from healthy 3-year-old male sika deer, and the antlers were sealed with starch. The cleaning process involved an ultrasonic cleaner with a frequency of 30kHz and a power of 200W, lasting 12 minutes at a temperature of 24℃. The drying process took 2.5 hours at 24℃. The first freezing was done with liquid nitrogen for 50 minutes, followed by immersion in anhydrous ethanol for 30 minutes. The second freezing was also done with liquid nitrogen for 2.5 hours.

[0074] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 25°C to obtain a mixture. The freeze-drying is carried out until the moisture content is 8%. The freeze-drying equipment is a freeze dryer with a working pressure of 0.8 MPa and the freeze-drying time is 6 hours. The pulverized powder is pulverized to 85% of the components with a particle size greater than 400 mesh. The mass ratio of deer antler powder to water is deer antler powder: water = 1:7. In step (2), the time for freezing the deer antler powder after dissolving it in water is 72 hours and the freezing temperature is -30°C.

[0075] (3) Lactic acid bacteria and yeast are added to the mixture for fermentation. After fermentation, the mixture is crushed three times using a low-temperature ultra-high pressure continuous flow cell crusher to obtain deer antler slurry. The fermentation is a two-stage process: first, anaerobic fermentation is carried out by lactic acid bacteria, and then aerobic fermentation is carried out by yeast. The lactic acid bacteria include bifidobacteria and lactobacilli, and the inoculation amount of bifidobacteria is 2×10⁻⁶. 7 CFU / ml, the inoculum size of the lactobacillus is 1×10⁻⁶. 7 CFU / ml; the yeast strain is *Diplostomum cuspidatum*, and the inoculum size is 0.5 × 10⁻⁶. 7 CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.14% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.2% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 38℃, the fermentation time was 5.5 h, and the stirring speed was 70 rpm. For aerobic fermentation with yeast, the fermentation temperature was 30℃, the fermentation time was 4 h, the stirring speed was 50 rpm, and the oxygen content of the fermentation broth was 0.8 mg / L.

[0076] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 0.8 MPa for 7.5 hours, the deer antler extract is obtained with a moisture content of 0.6% and a particle size of less than 100 μm of 99.5%.

[0077] Example 6

[0078] A method for preparing deer antler extract includes the following steps:

[0079] (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, cut it into 3.5 mm slices, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices.

[0080] The fresh deer antlers were selected from healthy 4-year-old male sika deer. The sealing method included one of the following: starch sealing, chitosan powder sealing, flame baking sealing, or soldering iron sealing. Cleaning was performed using an ultrasonic cleaner with a frequency of 20kHz and a power of 150W, for 10 minutes at a temperature of 21℃. Drying took 2 hours at 21℃. The first freezing was done with liquid nitrogen for 35 minutes, followed by immersion in anhydrous ethanol for 22 minutes. The second freezing was also done with liquid nitrogen for 2 hours.

[0081] (2) The deer antler slices are further freeze-dried and then pulverized in an airflow ultrafine pulverizer to obtain deer antler powder. The deer antler powder is dissolved in ultrapure water and then frozen, and then restored to 22°C to obtain a mixture. The freeze-drying is carried out until the moisture content is 6%. The freeze-drying equipment is a freeze dryer with a working pressure of 1.0 MPa and the freeze-drying time is 7 hours. The pulverized powder is pulverized to a particle size greater than 400 mesh, and the mass ratio of deer antler powder to water is deer antler powder: water = 1:9. In step (2), the time for freezing the deer antler powder after dissolving it in water is 68 hours and the freezing temperature is -17°C.

[0082] (3) Lactic acid bacteria and yeast are added to the mixture for fermentation. After fermentation, the mixture is crushed twice using a low-temperature ultra-high pressure continuous flow cell crusher to obtain deer antler slurry. The fermentation is a two-stage process: first, anaerobic fermentation is carried out by lactic acid bacteria, and then aerobic fermentation is carried out by yeast. The lactic acid bacteria include bifidobacteria and lactobacilli, and the inoculation amount of bifidobacteria is 10 × 10⁻⁶. 6 The inoculum size of the lactobacillus was 5.0 × 10⁻⁶ CFU / ml. 6 CFU / ml; the yeast includes *Candida utilis* and *Candida spp.*, and the inoculum size of *Candida utilis* is 0.5 × 10⁻⁶. 5 The inoculum size of *CFU / ml* was 0.5 × 10⁻⁶ CFU / ml. 5CFU / ml; lactose was added to the mixture as a carbon source at a concentration of 0.18% of the mixture's mass. Milk protein was added to the deer antler powder solution as a nitrogen source at a concentration of 0.22% of the deer antler powder solution's mass. For anaerobic fermentation with lactic acid bacteria, the fermentation temperature was 35℃, the fermentation time was 6 hours, and the stirring speed was 75 rpm. For aerobic fermentation with yeast, the fermentation temperature was 38℃, the fermentation time was 3 hours, the stirring speed was 25 rpm, and the oxygen content of the fermentation broth was 0.5 mg / L.

[0083] (4) After the deer antler slurry is dried in an industrial-grade freeze dryer at 1 MPa for 8 hours, the deer antler extract has a moisture content of 0.8% and a particle size of less than 100 μm of 99.3%, and the deer antler extract is obtained.

[0084] Comparative Example 1

[0085] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 1 differs only in step (1), which does not involve a second freezing, while the rest of the preparation methods are exactly the same.

[0086] Comparative Example 2

[0087] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 2 differs only in step (1), where the slices are not immersed in anhydrous ethanol, while the rest of the preparation methods are exactly the same.

[0088] Comparative Example 3

[0089] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 3 differs only in step (2). The deer antler slices are freeze-dried and then not pulverized. The rest of the preparation methods are exactly the same.

[0090] Comparative Example 4

[0091] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 4 differs only in step (2), where the deer antler powder is dissolved in ultrapure water and then not frozen. The rest of the preparation methods are exactly the same.

[0092] Comparative Example 5

[0093] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 5 differs only in step (3). It uses only lactic acid bacteria for anaerobic fermentation and does not add yeast for aerobic fermentation. The rest of the preparation methods are exactly the same.

[0094] Comparative Example 6

[0095] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 6 differs only in step (3). It uses yeast for aerobic fermentation and does not add lactic acid bacteria for anaerobic fermentation. The rest of the preparation methods are exactly the same.

[0096] Comparative Example 7

[0097] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 7 differs only in step (3). After fermentation, a non-low temperature ultra-high pressure continuous flow cell disruptor is used for crushing, and airflow ultra-micro pulverization is used. The rest of the preparation methods are exactly the same.

[0098] Comparative Example 8

[0099] Compared with the preparation method of deer antler extract in Example 1, Comparative Example 8 uses a traditional process to prepare deer antler extract, which includes boiling, baking, air drying, slicing, and ultra-fine pulverization. For details, please refer to [Li Heping, Wang Chunsheng. Ecological Deer Farming [M]. Beijing: China Agriculture Press, 2011].

[0100] Performance test examples and comparative examples of antler extract index detection

[0101] Testing standards:

[0102] Total protein content was determined using an automated Kjeldahl nitrogen analyzer, following the method outlined in GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Food.

[0103] The total amount of free amino acids was determined by adding pure water at a mass ratio of 1:10, stirring for 15 min, centrifuging at high speed, filtering the supernatant through 0.45 μm, and then determining it by formaldehyde titration.

[0104] Water-soluble total polysaccharides were prepared by water extraction-alcohol precipitation and determined by sulfuric acid-carbazole method;

[0105] Total fat content was determined by Soxhlet extraction, following the method specified in GB 5009.6-2016 National Food Safety Standard - Determination of Fat in Food.

[0106] The total amount of nucleosides was determined by ultra-high performance liquid chromatography (UPLC), and the total amounts of cytosine, uracil, adenine, guanine, hypoxanthine, xanthine, uridine, thymine, inosine, guanosine, adenosine, 2′-deoxyguanosine, and β-thymidine were statistically analyzed.

[0107] Moisture content was determined using a moisture meter, following the method specified in GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food.

[0108] Ash content was determined by muffle furnace ignition, following the method specified in GB5009.4-2016 National Food Safety Standard - Determination of Ash Content in Food.

[0109] The test results are shown in Table 1-2.

[0110] Table 1

[0111]

[0112] Table 2

[0113]

[0114] As shown in Tables 1-2, the deer antler extract prepared in the embodiments of the present invention has very high contents of total protein, total water-soluble amino acids, total water-soluble polysaccharides, total fat, and total nucleosides, especially higher than that obtained by traditional methods.

[0115] Application Test 1: Acute Oral Toxicity Test

[0116] Test method:

[0117] KM mice were pre-fed in the barrier environment animal room of our laboratory for 3 days to acclimatize to the environment. Before the experiment, KM mice were fasted overnight, but water intake was not restricted. A single-dose limited administration method was used, with an oral gavage dose of 5000 mg / kg body weight and an aqueous solution volume of 2 mL / 100g. Animal weight was measured and recorded before administration, and fasting continued for 3 hours after administration. Individual and comprehensive records were kept for each animal after administration. On the first day after administration, the toxic symptoms and mortality of KM mice were observed regularly, and thereafter, careful examination was performed daily. The observation period was 14 days. Surviving KM mice were weighed weekly during the observation period. At the end of the observation period, surviving KM mice were weighed, euthanized, and subjected to autopsy. Gross anatomical examination was performed on the KM mice, and all gross pathological changes were recorded. Histopathological examination was performed on organs from dead animals, animals that survived for more than 24 hours, and animals with gross pathological changes.

[0118] Experimental results:

[0119] No poisoning symptoms or deaths were observed in KM mice within 14 days of exposure; the average weight of both male and female animals remained normal. At the end of the observation period, gross anatomical examination of the test animals also revealed no abnormalities. Therefore, the acute oral LD50 of this test substance in KM mice is >5000 mg / kg•bw.

[0120] Table 3

[0121]

[0122]

[0123] Application Test 2: Superoxide Dismutase (SOD) Activity Assay

[0124] The superoxide dismutase (SOD) activity assay used the pyrogallol auto-oxidation method. The amount of SOD required to inhibit the pyrogallol auto-oxidation rate by 50% at 25℃ was defined as one activity unit.

[0125] Principle: Under alkaline conditions, pyrogallol undergoes auto-oxidation. The activity of SOD can be determined based on its ability to inhibit the auto-oxidation of pyrogallol.

[0126] Reagents: 0.1 mol / L Tris-HCl-EDTA buffer (pH 8.2): Dissolve 1.2114 g Tris and 37.2 mg EDTA in 62.4 mL / L hydrochloric acid solution, and bring the volume to 100 mL with distilled water. 45 mmol / L pyrogallol solution: Weigh 56.7 mg pyrogallol and dissolve it in 10 mmol / L hydrochloric acid solution, and bring the volume to 100 mL. 10 mmol / L hydrochloric acid solution, 0.200 mg / mL superoxide dismutase (SOD), distilled water.

[0127] Instruments: UV-Vis spectrophotometer, precision pH meter (0.01pH), 10ml colorimetric tubes.

[0128] Sample preparation: Add buffer solution and double-distilled water to a test tube, keep it at 25℃ for 20 min, then add a certain amount of 0.200 mg / ml superoxide dismutase, followed by pyrogallol preheated at 25℃ (10 mmol / L hydrochloric acid was used instead of control tube), shake well quickly, and immediately pour into a cuvette. Measure the absorbance every 30 s at a wavelength of 325 nm.

[0129] The test results are shown in Table 4.

[0130] Table 4

[0131]

[0132] As can be seen from the table above, the content of superoxide dismutase (SOD) in the deer antler extract prepared in the examples is much higher than that in the deer antler extract prepared in the comparative examples.

[0133] Application Test 3 Antioxidant Activity

[0134] (1) Investigation on DPPH free radical inhibition

[0135] DPPH is a very stable nitrogen free radical. Its methanol or ethanol solution is deep purple-red. When a free radical scavenger is added to the DPPH free radical solution, the deep purple DPPH free radical is reduced to the yellow DPPH-H non-free radical form. The degree of fading is quantitatively related to the number of electrons accepted, so it can be quantitatively analyzed by changes in absorbance.

[0136] Accurately weigh the samples from the examples and comparative examples, add 99 g of double-distilled water, and incubate at 37°C with shaking for 1 hour. Filter the filtrate through a 0.45 μm microfiltration membrane and set aside. Take 100 μL of the sample solution, add 100 μL of 0.05% DPPH ethanol solution, mix well, and react in a dark chamber for 1 hour. Measure the absorbance at 517 nm, repeating three times and taking the average value. Use 100 μL of distilled water as a control; use 100 μL of anhydrous ethanol plus 100 μL of the corresponding sample as a blank. Calculate the DPPH free radical scavenging rate.

[0137] Clearance rate (%) = [1-(A)] 样品 -A 空白 ) / A 对照 ]×100%

[0138] The test results are shown in Table 5.

[0139] Table 5

[0140]

[0141] The experimental results show that the DPPH free radical scavenging rate of the deer antler extract prepared in the embodiments of the present invention is higher than 80%; the scavenging rate of the comparative example is slightly lower, while the scavenging rate of commercially available processed deer antler slices is the lowest, at only 20.17%. Therefore, the processing method adopted in the present invention prevents the loss of nutrients and functional activity of deer antler during processing, resulting in high biological activity and strong ability to scavenge DPPH free radicals.

[0142] (2) Investigation on the inhibition of hydroxyl radicals

[0143] Use Fe 3＋ - EDTA, ascorbic acid, and H₂O₂ undergo a Fenton reaction to generate hydroxyl radicals (·OH), which can degrade deoxyribose. In an acidic environment, the degradation products can react with TBA (thiobarbituric acid) to form a pink substance, which can be detected colorimetrically to determine the degree of deoxyribose degradation. By analyzing the oxidation of deoxyribose molecules by ·OH radicals, it can be determined whether the sample has the effect of inhibiting hydroxyl radical (·OH) formation.

[0144] Accurately weigh the products from the example and comparative examples, add 99g of double-distilled water, and shake at 37°C for 1 hour. Filter the filtrate through a 0.45μm microfiltration membrane and set aside for use.

[0145] Add 0.40 mL of 50 mmol / L pH 7.5 phosphate buffer, 1.04 mmol / L EDTA solution, 1 mmol / L FeCl3, 0.10 mL of a sample of a certain concentration (distilled water was used for the control tube), 0.10 mL each of 60 mmol / L deoxyribose (blank) and 10 mmol / L H2O2 to a test tube and mix well. Incubate at 37°C for 1 hour, then immediately add 1.0 mL of 20% TCA (trichloroacetic acid) to terminate the reaction. Add 1.0 mL of 0.8% TBA (thiobarbituric acid) colorimetric reagent, and react in a boiling water bath for 15 minutes. Immediately cool with ice water and measure the absorbance at 532 nm to calculate the clearance rate.

[0146] Clearance rate (%) = [1 - (A)] 样品 -A 空白 ) / (A 对照 -A 空白 )]×100%

[0147] The test results are shown in Table 6.

[0148] Table 6

[0149]

[0150] The experimental results show that the deer antler extract prepared in the embodiments of the present invention has a better scavenging rate of hydroxyl radicals (·OH), the scavenging rate of the comparative embodiments is slightly lower, and the scavenging rate of commercially available processed deer antler slices is the lowest.

[0151] Application Test 4: Anti-fatigue animal test

[0152] Fatigue is a complex physiological and biochemical process, a normal physiological phenomenon that inevitably occurs when mental or physical exertion reaches a certain stage. It signifies a temporary decline in the body's original working capacity and may also be a precursor to disease. The Fifth International Conference on Exercise Biochemistry in 1982 standardized the concept of fatigue as: the inability of the body's physiological processes to sustain its function at a specific level or the inability of organs to maintain a predetermined exercise intensity. From the central nervous system to skeletal muscle cells and intracellular metabolic processes, any change in any link or process can cause fatigue. According to a WHO survey, more than 35% of the global population is in a state of fatigue, with the figure reaching 60% among middle-aged men. Fatigue can lead to reduced athletic ability, decreased work efficiency, increased errors and accidents, and decreased combat effectiveness. If fatigue is not addressed promptly, it can accumulate and lead to overwork, resulting in chronic fatigue syndrome (CFS) and overtraining syndrome, causing endocrine disorders, weakened immunity, and even organic diseases, threatening human health. The anti-fatigue function of the deer antler extract of this invention was evaluated using animal testing.

[0153] The drug was administered to mice via gavage. The experimental method was based on the study conducted by Wang Wenlong, Research on Deer Antler Tablets for Improving Immunity and Combating Fatigue [D], Jilin University, 2018. 0.5, 1.0, and 3.0 g of the deer antler extract sample prepared in Example 1 were diluted with distilled water to prepare 100 mL of reagent as low, medium, and high dose groups, respectively. Distilled water served as a blank control group. Each group consisted of ≥30 mice, and the gavage dose for each group was 0.2 mL / 10 g BW d, administered continuously for 30 days. On day 30, a weighted swimming test was conducted. The mice were placed in a swimming tank with a water depth of approximately 30 cm at room temperature. A weight equal to 5% of the mouse's body weight was fixed at the base of the tail to accelerate the experiment. The time (min) from the start of swimming to sinking was recorded.

[0154] The results are shown in Table 7.

[0155] Table 7

[0156]

[0157] The experimental results show that after administering different doses of the deer antler extract sample prepared in Example 1 to experimental mice by gavage for 30 days, the swimming time of the medium and high dose groups was longer than that of the blank control group, and the differences were significant (P<0.05), indicating that the sample prolongs the swimming time of experimental mice under load.

[0158] Using a moderate dose as the standard, the effects of different deer antler extract samples on experimental mice were investigated, with ≥10 mice in each group.

[0159] The results are shown in Table 8.

[0160] Table 8

[0161]

[0162] Experimental results show that, when fed with a moderate dose to experimental mice, the deer antler extract prepared in the example is significantly better than the deer antler extract prepared in the comparative example in improving immunity and anti-fatigue in experimental mice.

[0163] Application Test 5: In vitro skin barrier protection and anti-inflammatory test

[0164] Skin contact with highly irritating test substances or ultraviolet radiation can cause acute clinical damage to the skin barrier, leading to dry skin and erythema. Anionic surfactants, such as sulfadiazine (SLS), are amphiphilic (both hydrophilic and lipophilic), and at high concentrations, they can damage the skin barrier, particularly its lipid components and cell membranes. Tissue morphology analysis, performed after H&E staining, reveals changes in the skin barrier under different treatment conditions. For example, SLS damage weakens the skin barrier and reduces the thickness of the viable cell layer, while treatment with the active ingredient improves this damage. Corneocyte occlusion (CE) forms the structural basis of the skin's permeability barrier. Lorheological protein (LOR) is a key component in CE assembly, accounting for 80% of the protein and playing a crucial reinforcing role. Decreased LOR protein content is a major factor weakening skin barrier function. Filamentin (FLG) is also a key component in CE assembly. Besides being a structural component of the skin barrier, FLG can be hydrolyzed by Caspase-14 to form natural moisturizing factors. Therefore, the protective ability of the sample on the skin can be assessed by observing changes in the morphology of the model tissue, lobe rhinol (LOR), and filaggrin (FLG) content after drug administration.

[0165] The most typical clinical symptom of skin barrier damage is skin redness, indicating an inflammatory response. Inflammatory factors and mediators are relevant indicators of inflammatory responses. SLS stimulation leads to a large secretion of inflammatory factors, while the active ingredient significantly reduces these factors, thus achieving the effect of repairing skin barrier damage. IL-1α is a pro-inflammatory factor, a non-hydrophobic polypeptide that can only be released extracellularly after cell membrane damage. The decrease in inflammatory factor levels after the application of the analyte indicates a soothing effect. Prostaglandin PGE2 is a metabolite of arachidonic acid, mainly synthesized through cyclooxygenase. PGE2 can dilate blood vessels, leading to erythema, and can also induce clinical symptoms of inflammatory pain. The decrease in PGE2 levels after the application of the analyte indicates a soothing effect. Therefore, the anti-inflammatory capacity of a sample can be evaluated by observing changes in the levels of the pro-inflammatory factor IL-1α and the inflammatory mediator PGE2 after drug administration.

[0166] This test used a 3D epidermal skin model (EpiKutis®). The samples were 1g each of Example 1 and Comparative Example 8, accurately weighed, and 99g of double-distilled water was added. The mixture was shaken at 37°C for 1 hour, and the filtrate was filtered through a 0.45μm microfiltration membrane for later use.

[0167] The test results are shown in Table 9.

[0168] Table 9

[0169]

[0170] From the table above and Figures 1 - 3 The experimental results show that the deer antler extract prepared in this invention has a significant repairing effect on the skin barrier and can inhibit the release of inflammatory factors.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing deer antler extract, characterized in that, Includes the following steps: (1) After sealing the cut surface of the unossified fresh deer antler, wash it, dry it, freeze it for the first time, slice it, immerse it in anhydrous ethanol, dry it, freeze it for the second time, and obtain deer antler slices. (2) The deer antler slices are freeze-dried and then pulverized to obtain deer antler powder. The deer antler powder is dissolved in water and then frozen, and then restored to 15-25℃ to obtain a mixture. (3) Add lactic acid bacteria and yeast to the mixture for fermentation. After fermentation, crush the mixture using a low-temperature ultra-high pressure continuous flow cell crusher to obtain deer antler slurry. The fermentation is a two-stage process, with anaerobic fermentation by lactic acid bacteria followed by aerobic fermentation by yeast. The lactic acid bacteria are composed of Bifidobacterium and Lactobacillus, and the yeast is at least one of Candida utilis and Trichoderma spp. (4) After freeze-drying the deer antler slurry, the deer antler extract is obtained.

2. The method for preparing deer antler extract as described in claim 1, characterized in that, In step (1), the cleaning is performed using an ultrasonic cleaner for 5-15 minutes at a temperature of 15-30°C; the drying time is 1-3 hours at a temperature of 20-25°C; the first freezing is performed using liquid nitrogen for 30-60 minutes, followed by immersion in anhydrous ethanol for 15-30 minutes; and the second freezing is performed using liquid nitrogen for 1-3 hours.

3. The method for preparing deer antler extract as described in claim 1, characterized in that, In step (2), the antler powder is freeze-dried until the moisture content is ≤15%, and the powder is pulverized until the particle size is greater than 400 mesh >70%. The mass ratio of the antler powder to water is antler powder:water = 1:(5-10). In step (2), the freezing time after the antler powder is dissolved in water is 48-72h, and the freezing temperature is -10~-30℃.

4. The method for preparing deer antler extract as described in claim 1, characterized in that, In step (3), the inoculation amount of Bifidobacterium is 3 × 10⁻⁶. 5 -10×10 7 CFU / ml, the inoculum size of the lactobacillus is 1.5 × 10⁻⁶. 5 -5.0×10 7 CFU / ml; the inoculum size of the yeast is 1.0 × 10⁻⁶. 5 -1.0×10 7 CFU / ml.

5. The method for preparing deer antler extract as described in claim 4, characterized in that, The Bifidobacterium is at least one of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, and Bifidobacterium bifidum; the Lactobacillus is at least one of Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus breve, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus plantarum.

6. The method for preparing deer antler extract as described in claim 1, characterized in that, In step (3), when lactic acid bacteria undergo anaerobic fermentation, the fermentation temperature is 20-45℃, the fermentation time is 3-8h, and the stirring speed is 50-100rpm; when yeast undergoes aerobic fermentation, the fermentation temperature is 30-42℃, the fermentation time is 2-6h, the stirring speed is 10-50rpm, and the oxygen content of the fermentation liquid is 0.5-1.0mg / L.

7. The method for preparing deer antler extract as described in claim 1, characterized in that, In step (4), the freeze-drying time is 6-8 hours, the moisture content of the deer antler extract is ≤1%, and the component with a particle size of less than 100 μm is >99%.

8. Deer antler extract prepared by the method for preparing deer antler extract according to any one of claims 1-7.

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