Preparation process of fresh animal medicine

Abstract

The application relates to the technical field of animal medicine preparation, and discloses a fresh animal medicine preparation process, which comprises the following steps: step (1), cleaning treatment: removing non-medicinal parts of fresh animal medicine, cutting and cleaning; step (2), low-temperature freezing storage: freezing and storing the cleaned fresh animal medicine in an environment with a temperature of-15 DEG C to-25 DEG C; step (3), homogenate treatment: cutting the frozen fresh animal medicine, adding an equal amount of water to prepare a homogenate under the condition that the ambient temperature is not higher than 19 DEG C; and step (4), freeze-thaw extraction. Through the technical scheme of low-temperature freezing storage and repeated freeze-thaw extraction, the physical damage of cell membranes is realized through the formation and dissolution of ice crystals, active ingredients such as proteins and enzymes in the fresh animal medicine are fully released, protein denaturation and activity loss caused by heating are avoided, the extraction rate of effective components is significantly improved, and the problem that the natural activity of fresh animal medicine cannot be preserved by traditional methods is solved.

Description

Technical Field

[0001] This invention relates to the field of animal drug preparation technology, specifically a process for preparing fresh animal drugs. Background Technology

[0002] Traditional Chinese medicine (TCM) holds an important position in traditional medicine due to its unique efficacy and abundant resources. Among them, animal-based medicines, because of their similarity to human structures and unique bioactive components, are considered "products with flesh and blood," playing an irreplaceable role in treating acute and severe illnesses and complex internal medicine conditions. Animal-based medicines are rich in active substances such as proteins, enzymes, and polypeptides, which directly affect the efficacy of the drugs. However, fresh animal-based medicines, due to their high protein and moisture content, are extremely prone to spoilage, and their processing and preservation have always been technical challenges in the industry.

[0003] Currently, the processing and preservation of fresh animal-derived medicines mostly employ methods such as roasting and air-drying. While these processes can extend shelf life, they often damage the active ingredients. For example, high-temperature boiling can lead to protein denaturation and peptide degradation, significantly reducing biological activity; during drying, some key components may become ineffective due to oxidation. Furthermore, although traditional roasting and air-drying processes avoid high-temperature treatment, their moisture removal efficiency is low, resulting in high residual moisture in the product, which can easily affect the stability of the finished product. Existing technologies are currently unable to efficiently extract the active ingredients of fresh animal-derived medicines while simultaneously ensuring high purity and long-term stability of the finished product.

[0004] A major problem with existing technologies is that the processing techniques for fresh animal-derived medicines struggle to balance the extraction efficiency of active ingredients with the stability of the finished product. Insufficient cell disruption during extraction leads to inadequate release of key components, resulting in low levels of active ingredients. Furthermore, incomplete moisture removal during the drying stage makes the product prone to spoilage and deterioration during storage. Therefore, there is an urgent need for a preparation process that can efficiently extract the active ingredients from fresh animal-derived medicines while ensuring a low-moisture, high-stability final product. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a fresh animal drug preparation process that solves the problems of low extraction efficiency of active ingredients and poor product stability in existing fresh animal drug processing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a process for preparing fresh animal-derived medicine, comprising the following steps:

[0007] Step (1) Cleaning: Remove non-medicinal parts from the fresh animal medicine, cut it into pieces and clean it;

[0008] Step (2) Low-temperature freezing storage: Place the cleaned fresh animal medicine in an environment of -15℃ to -25℃ for freezing storage;

[0009] Step (3) Homogenization: Cut the frozen fresh animal medicine into pieces and add an equal amount of water to make a homogenate at an ambient temperature not higher than 19°C;

[0010] Step (4) Freeze-thaw extraction: After homogenizing the material, freeze it at -25℃±2℃ for 20-24 hours, and then thaw it at 37℃±1℃ for 20-24 hours. Repeat this process 2-4 times.

[0011] Step (5) Separation: The frozen and thawed materials are separated by low-speed centrifugation, high-speed centrifugation and ultrafiltration in sequence;

[0012] Step (6) Freeze-drying: The separated liquid is freeze-dried at low temperature, wherein the pre-freezing temperature is below -35℃, the sublimation temperature is from -20℃ to -5℃, and the drying temperature is 35℃±2℃.

[0013] Preferably, the homogenization process in step (3) includes:

[0014] Add distilled or sterile water at a 1:1 ratio during homogenization;

[0015] Homogenization time is 20-40 minutes.

[0016] Preferably, the freeze-thaw extraction in step (4) includes:

[0017] During the freeze-thaw process, the material is frozen for 20-24 hours under freezing conditions;

[0018] During the melting stage, the material is melted in a water bath environment for 20-24 hours, and the freeze-thaw cycle is repeated 3 times.

[0019] Preferably, the low-speed centrifugation in step (5) includes the following conditions:

[0020] The centrifuge speed is 3000-7000 rpm, and the time is 10-20 minutes;

[0021] The centrifugation was performed three times, with the flow rates of the liquid controlled at 15 L / min, 12 L / min, and 10 L / min, respectively.

[0022] Preferably, the high-speed centrifugation in step (5) includes the following conditions:

[0023] The high-speed centrifuge operates at 8000-12000 rpm for 5-10 minutes.

[0024] The flow rate of the medicine solution is less than 5L / min.

[0025] Preferably, the ultrafiltration separation in step (5) includes:

[0026] Use ultrafiltration membranes with a molecular weight cutoff of less than 50,000;

[0027] The filtration flow rate is 1-3L / min, and the filtration temperature is controlled below 19℃.

[0028] Preferably, the freeze-drying in step (6) includes the following conditions:

[0029] Pre-freezing time is 1-3 hours, and pre-freezing temperature is below -35℃;

[0030] The sublimation drying time is 6-12 hours, and the vacuum degree is below 35 Pa.

[0031] The final product has a moisture content of ≤6% during the drying process.

[0032] Preferably, the content of volatile alkaline substances in the fresh animal medicine during the cleaning step, calculated as nitrogen, does not exceed 33 mg / 100g.

[0033] Preferably, all processes in steps (3) to (6) are carried out in an environment where the temperature is not higher than 19°C.

[0034] This invention provides a process for preparing fresh animal-derived medicines. It has the following beneficial effects:

[0035] 1. This invention employs a low-temperature freezing storage and repeated freeze-thaw extraction technique. The formation and dissolution of ice crystals physically disrupt cell membranes, allowing for the full release of active ingredients such as proteins and enzymes from fresh animal-derived medicines. Compared to existing high-temperature decoction methods, this avoids protein denaturation and activity loss caused by heating, significantly improving the extraction rate of effective components and overcoming the shortcomings of traditional methods in preserving the natural activity of fresh animal-derived medicines.

[0036] 2. This invention combines multi-stage centrifugation and ultrafiltration separation technologies to refine the extraction of small-molecule active ingredients. Through multi-stage centrifugation and membrane filtration, the proportion of effective ingredients with a molecular weight below 10kDa reaches over 98%, far exceeding the purification effect of traditional filter screens, filter cloths, or filter papers. Existing technologies result in low purity of extracted active ingredients and numerous impurities, making it difficult to meet the requirements of high-efficiency drug preparation. This invention completely solves this problem, resulting in more stable drug quality.

[0037] 3. This invention achieves controllable high-efficiency component extraction and separation through a clearly defined design of low-temperature freezing, freeze-thaw cycles, and temperature parameters. Existing technologies often rely on experience-based operations, resulting in poor process repeatability and affecting extraction efficiency and product consistency. This invention, through a systematic process design, overcomes the shortcomings of traditional processes, such as technical instability and difficulty in standardizing efficacy, providing a completely new solution for the modern preparation of fresh animal medicines. Attached Figure Description

[0038] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Please see the appendix Figure 1 This invention provides a process for preparing fresh animal medicine, comprising:

[0041] Step 1: Raw material preparation

[0042] Cleaning and cutting: The fresh animal medicine is initially processed, including cleaning to remove impurities, cutting off non-medicinal parts, and cutting the medicine into small pieces of 3-5cm to facilitate subsequent processing.

[0043] Testing: Test the content of volatile alkaline substances (calculated as nitrogen) in the medicinal materials to ensure that it is ≤33mg / 100g, so as to guarantee the quality of raw materials and the freshness of medicinal components.

[0044] During storage, the decomposition and spoilage of proteins in fresh animal-derived medicines generate volatile alkaline substances. This detection step can promptly remove substandard raw materials, preventing them from affecting the extraction efficiency and drug stability of subsequent processes. Simultaneously, cutting the medicinal materials into pieces increases the contact area between the herbs and the freezing medium and subsequent processing equipment, enhancing freezing and extraction effects.

[0045] Step 2: Low-temperature freezing storage

[0046] After cleaning, store the medicinal materials in a frozen environment at -15℃ to -25℃ for no more than 30 days.

[0047] Low temperatures can inhibit the growth and metabolic activities of microorganisms in fresh animal-derived medicines, while effectively slowing down the degradation of proteins and enzymes, thus preserving their natural activity. Furthermore, the formation of ice crystals during freezing provides a physical basis for subsequent freeze-thaw extraction. The formation of ice crystals causes mechanical forces to act on cell membranes and cell walls, creating favorable conditions for the release of active ingredients.

[0048] Step 3: Repeated freeze-thaw extraction and homogenization: Add distilled water or sterile water to the frozen medicinal materials at an ambient temperature ≤19℃ to make a homogenate at a ratio of 1:1. The homogenization time is 20-40 minutes.

[0049] Freeze-thaw cycle: The homogenized material is frozen at -25℃±2℃ for 20-24 hours, and then thawed at 37℃±1℃ for 20-24 hours.

[0050] Repeat the freeze-thaw process 2-4 times.

[0051] Cell structure damage: When frozen, water forms ice crystals, and the expansion in volume causes the cell membrane and cell wall to rupture under mechanical force.

[0052] As the number of freeze-thaw cycles increases, the repeated formation and thawing of ice crystals further weakens the integrity of the cell membrane, thereby releasing active components such as proteins, enzymes, and amino acids.

[0053] Salt concentration changes: During freezing, the concentration of salts in the liquid increases, leading to changes in cell membrane permeability and further promoting the extravasation of intracellular components.

[0054] Gentle extraction: Freeze-thaw extraction completely avoids the damage to active ingredients caused by high-temperature cooking, and helps maintain the natural structure and functional activity of proteins and enzymes.

[0055] Step 4: Graded separation by low-speed centrifugation: The frozen and thawed material is centrifuged three times at low speed using a plate centrifuge, with the speed controlled at 3000-7000 rpm and the time at 10-20 minutes respectively.

[0056] The centrifugal flow rate was controlled at 15 L / min (first time), 12 L / min (second time), and 10 L / min (third time).

[0057] High-speed centrifugation: The supernatant after low-speed centrifugation is placed in a tubular centrifuge and subjected to two high-speed centrifugations:

[0058] The first rotation speed is 8000-10000 rpm, the flow rate is ≤5L / min, and the time is 5-10 minutes;

[0059] The second rotation speed is 10000-12000 rpm, the flow rate is ≤2.5L / min, and the time is 5-10 minutes.

[0060] Ultrafiltration separation: The liquid after high-speed centrifugation is filtered through an ultrafiltration membrane with a molecular weight cutoff of less than 50,000, with the filtration temperature controlled at ≤19℃ and the filtration flow rate at 1-3L / min.

[0061] Low-speed centrifugation gradually removes large particles and impurities from the drug solution;

[0062] High-speed centrifugation further separates small particulate impurities and protein polymers.

[0063] Molecular screening: Ultrafiltration membranes use a screening mechanism to retain only small molecules with a molecular weight of less than 50,000, including small molecules of proteins, peptides and amino acids, which have better pharmacological activity and bioavailability.

[0064] To avoid heat damage: the entire process is carried out at low temperatures to ensure the stability of the extract.

[0065] Step 5: Low-temperature freeze-drying pre-freezing stage: Place the separated drug solution in a freeze dryer, with a pre-freezing temperature ≤ -35℃ and a time of 1-3 hours.

[0066] Sublimation drying: Under vacuum conditions of <35Pa, sublimation drying is carried out at a temperature controlled between -20℃ and -5℃ for 6-12 hours.

[0067] Analysis and drying:

[0068] The temperature is gradually increased to 35℃±2℃ and maintained for 6 hours until the moisture content of the medicinal materials is ≤6%.

[0069] Drying mechanism: Freeze-drying directly converts the water in the drug solution from solid to gaseous state through sublimation, avoiding protein denaturation and degradation of active substances that may be caused by high-temperature drying.

[0070] Enhanced stability: Reducing the moisture content to below 6% helps improve the stability of the product and extend its shelf life.

[0071] Integrity protection: Under low-temperature drying conditions, the proteins, enzymes and small molecules in fresh animal medicines can maintain their natural structure, thereby preserving the bioactivity of the medicinal materials.

[0072] This process overcomes the loss of active ingredients caused by high-temperature treatment in traditional processes by combining low-temperature freezing, repeated freeze-thaw extraction, and fractional separation techniques with low-temperature freeze-drying. Each technical step is rationally designed based on scientific principles such as cell structure, physical separation, and molecular stability, achieving efficient extraction and preservation of active ingredients from fresh animal medicines.

[0073] Example 1: Preparation of Fresh Gecko as a Medicinal Herb

[0074] Step 1: Raw material preparation

[0075] Fresh gecko medicinal materials were collected, and non-medicinal parts were removed. The geckos were thoroughly rinsed with cold water. The pieces were cut into approximately 4cm x 4cm cubes and weighed using an electronic scale. Each batch consisted of 2kg of raw material. The content of volatile alkaline substances (calculated as nitrogen) was tested and found to be 28mg / 100g, which met the requirements.

[0076] Step 2: Low-temperature freezing storage

[0077] Place the chopped gecko raw material in a low-temperature freezer at -20℃. The storage time is set at 10 days. During this period, check the ice layer coverage to ensure even freezing.

[0078] Step 3: Homogenization

[0079] The frozen gecko blocks were placed in a bone mortar and pestle mill, and cold distilled water was added in a 1:1 ratio. The milling speed was set to 800 rpm, and the homogenization time was 30 minutes. The homogenized material was a uniform paste with no obvious particles.

[0080] Step 4: Freeze-thaw extraction

[0081] The homogenate was placed in a freezing device at -25°C for 24 hours. After freezing, it was placed in a water bath at 37°C ± 1°C for 22 hours to thaw. This freeze-thaw cycle was repeated three times until the homogenate was significantly diluted and the protein concentration showed a significant increase.

[0082] Step 5: Low-speed and high-speed centrifugation

[0083] The frozen-thawed liquid sample was placed in a plate centrifuge and centrifuged at 5000 rpm for 15 minutes to separate coarse particles. The supernatant was then collected. Subsequently, it was transferred to a tubular centrifuge and centrifuged at high speed of 10000 rpm for 8 minutes to separate out small impurity particles.

[0084] Step 6: Ultrafiltration separation

[0085] The liquid after high-speed centrifugation was passed through an ultrafiltration membrane with a molecular weight cutoff of 50,000, maintained at 18°C, and a flow rate of 2.5 L / min. The extract was clear and transparent, with a significantly increased proportion of small molecule proteins.

[0086] Step 7: Freeze-drying

[0087] The ultrafiltration extract was transferred to a freeze dryer, with a pre-freezing temperature set at -35℃ for 2 hours. Sublimation drying was then carried out under a vacuum of 30 Pa at -10℃ for 10 hours. The final desorption drying was performed at 35℃ for 5 hours, yielding a dry powder with a moisture content of 5.8%.

[0088] Example 2: Preparation of fresh earthworm as a medicinal material

[0089] Step 1: Raw material preparation

[0090] Fresh earthworm herbs were selected and rinsed to remove surface mud and impurities. Surface residue was cleaned with a peeler, and the herbs were cut into sections approximately 5cm in length. Preliminary testing showed that the content of volatile alkaline substances was 30mg / 100g.

[0091] Step 2: Low-temperature freezing storage

[0092] The earthworm medicinal materials were placed in batches into a freezing device, with the freezing temperature set to -18℃ for 14 days.

[0093] Step 3: Homogenization

[0094] After being frozen, the earthworms were removed and processed using a high-speed homogenizer. An equal amount of cold distilled water was added, and the homogenization time was set to 25 minutes. The final homogenized material was a uniform paste with no suspended solid particles.

[0095] Step 4: Freeze-thaw extraction

[0096] The homogenized material was transferred to a freezer at -23°C for 20 hours. It was then removed and thawed in a 37°C water bath for 20 hours. After four freeze-thaw cycles, the protein content reached its peak.

[0097] Step 5: Centrifugation

[0098] The extract was transferred to a plate centrifuge and centrifuged at low speed (4000 rpm) for 12 minutes. The supernatant was then centrifuged again at 6000 rpm for 10 minutes. Finally, it was transferred to a high-speed centrifuge at 11000 rpm for 6 minutes to completely remove high molecular weight impurities.

[0099] Step 6: Membrane filtration

[0100] The supernatant was passed through an ultrafiltration device using a membrane with a molecular weight cutoff of less than 50,000, at a flow rate of 3 L / min and a temperature maintained at 17°C. The filtered liquid was clear, and its main components were small-molecule proteins and amino acids.

[0101] Step 7: Freeze-drying

[0102] The extract was transferred to a freeze-drying apparatus, pre-freezing at -30°C for 1.5 hours. During sublimation drying, a vacuum of 28 Pa was applied, the sublimation temperature was set to -15°C, and the time was 8 hours. After drying, the product had a moisture content of 5.2%.

[0103] Example 3: Preparation of Fresh Snake (Golden Flower Snake) as a Traditional Chinese Medicine

[0104] Step 1: Raw material preparation

[0105] The internal organs and head of the fresh white-flowered snake were removed, and any residue was washed away before cutting it into 3cm long pieces. The content of volatile alkaline substances was found to be 32mg / 100g.

[0106] Step 2: Low-temperature freezing storage

[0107] The freezing temperature for medicinal materials is set to -25℃, and the storage time is 7 days.

[0108] Step 3: Homogenization

[0109] Take frozen white-flowered snake and add cold distilled water at a ratio of 1:0.8 to form a homogenate, which is then homogenized for 35 minutes. The resulting homogenate is fine and pale pinkish-white.

[0110] Step 4: Freeze-thaw extraction

[0111] The homogenate was frozen at -24°C for 24 hours. It was then thawed in a 36°C water bath for 21 hours. This freeze-thaw cycle was repeated three times.

[0112] Step 5: Low-speed centrifugation

[0113] The frozen and thawed liquid was placed in a low-speed centrifuge at 4500 rpm for 12 minutes. The supernatant was collected.

[0114] Step 6: High-speed centrifugation and ultrafiltration separation

[0115] The high-speed centrifuge operates at 10,000 rpm for 7 minutes. The supernatant is then passed through an ultrafiltration system to remove small molecules with a molecular weight below 50,000. The filtration temperature is 16℃.

[0116] Step 7: Freeze-drying

[0117] The pre-freezing temperature was -33℃ for 2 hours. The sublimation temperature was controlled at -12℃ for 9 hours, and the final desorption and drying temperature was 33℃. The resulting dry powder had a moisture content of 5.5%.

[0118] Example 4: Preparation of fresh Agkistrodon halys animal medicine

[0119] Steps 1-7 (same as the above technical steps, but adjust the number of freeze-thaw cycles to 4 times and the frozen storage time to 12 days).

[0120] Example 5: Preparation of Fresh Earthworm Animal Medicine

[0121] Steps 1-7 (same as the above technical steps, but adjust the high-speed centrifugation speed to 12000 rpm and the ultrafiltration separation temperature to 15℃).

[0122] Comparative Example 1:

[0123] Raw material preparation: Select fresh gecko medicinal materials, remove non-medicinal parts and rinse, cut into pieces approximately 4cm × 4cm in size. The content of volatile alkaline substances was tested to be 28mg / 100g. The preparation steps are the same as in Example 1.

[0124] Low-temperature frozen storage: The freezer temperature is set to -10℃, and the freezing time is 15 days.

[0125] Homogenization: Add distilled water to the frozen gecko blocks at a ratio of 1:0.5, and use a low-speed bone paste mill for 15 minutes to homogenize.

[0126] Freeze-thaw extraction: The homogenized material is frozen at -15°C for 12 hours, and then thawed in a 45°C water bath for 10 hours. The freeze-thaw cycle is performed only once.

[0127] Separation steps:

[0128] Low-speed centrifugation: Set the speed to 2000 rpm and the time to 10 minutes.

[0129] High-speed centrifugation: 6000 rpm for 5 minutes.

[0130] Ultrafiltration separation: Use a filter membrane with a molecular weight cutoff of 100,000, filter temperature of about 25°C, and flow rate of 5L / min.

[0131] Freeze-drying: The pre-freezing temperature is set to -20℃, the sublimation temperature to -5℃, the sublimation time to 6 hours, and the desorption drying temperature to 40℃.

[0132] Comparative Example 2:

[0133] Raw material preparation: Fresh earthworm medicinal material was taken and processed in the same way as in Example 2, with the length of the cut pieces being 5cm. The volatile alkaline substances test was qualified.

[0134] Low-temperature freezing storage: The freezing equipment temperature is set to -5℃, and the freezing time is 5 days.

[0135] Homogenization: The frozen earthworms were treated with a high-speed homogenizer, and cold water was added at a ratio of 1:1.5. The homogenization time was shortened to 10 minutes.

[0136] Freeze-thaw extraction: The homogenate was frozen at -10℃ for 6 hours, and then thawed in a 50℃ water bath for 8 hours. The freeze-thaw process was not repeated and was performed only once.

[0137] Separation steps

[0138] Low-speed centrifugation: 3000 rpm for 8 minutes.

[0139] High-speed centrifugation: 8000 rpm for 5 minutes.

[0140] Ultrafiltration separation: Use an ultrafiltration membrane with a molecular weight cutoff of 200,000, a flow rate of 4 L / min, and a filtration temperature of 22℃.

[0141] Freeze-drying: Pre-freeze temperature -15℃, time 1 hour. Sublimation temperature 0℃, sublimation time 5 hours, and desorption drying temperature set at 38℃.

[0142] Comparative Example 3:

[0143] Raw material preparation: Remove the internal organs and head of the fresh *Gynostemma pentaphyllum* snake, and cut it into sections using the same method as in Example 3. Each section should be approximately 3cm long. The content of volatile alkaline substances should meet the requirements.

[0144] Low-temperature freezing storage: The freezing equipment temperature is set to 0℃, and the freezing time is 3 days.

[0145] Homogenization: Add cold water to the homogenate at a ratio of 1:2 and process with a low-speed homogenizer, reducing the homogenization time to 5 minutes.

[0146] Freeze-thaw extraction: Freezing temperature -5℃, freezing time 4 hours. Thawing conditions: 55℃ water bath for 4 hours. The freeze-thaw process was not repeated; only one operation was performed.

[0147] Separation steps:

[0148] Low-speed centrifugation: 3500 rpm for 5 minutes.

[0149] High-speed centrifugation: 7000 rpm for 5 minutes.

[0150] Ultrafiltration separation: Use an ultrafiltration membrane with a molecular weight cutoff of 300,000, a filtration temperature of 25°C, and a flow rate of 6 L / min.

[0151] Freeze-drying: Pre-freeze temperature -10℃, time 0.5 hours. Sublimation temperature 5℃, sublimation time 4 hours. Desorption drying temperature set at 42℃.

[0152] Comparative Example 4:

[0153] Raw material preparation: The processing method for the Agkistrodon halys is the same as in Example 4. The internal organs and head are removed, and the snake is cut into 5cm segments. The volatile alkaline substances test results are satisfactory.

[0154] Low-temperature frozen storage: The freezing temperature is set to -8℃, and the freezing time is 8 days.

[0155] Homogenization: Add cold distilled water to the frozen Agkistrodon halys homogenate at a ratio of 1:0.8 and homogenize for 20 minutes.

[0156] Freeze-thaw extraction: Freezing temperature -12℃, freezing time 8 hours, thawing temperature 48℃, thawing time 12 hours. Only 2 freeze-thaw cycles were performed.

[0157] Separation steps

[0158] Low-speed centrifugation: 4000 rpm for 8 minutes.

[0159] High-speed centrifugation: 9000 rpm for 6 minutes.

[0160] Ultrafiltration separation: Use an ultrafiltration membrane with a molecular weight cutoff of 150,000, a filtration temperature of 20°C, and a flow rate of 5 L / min.

[0161] Freeze-drying: Pre-freeze temperature -25℃, time 1 hour. Sublimation temperature -10℃, sublimation time 8 hours, and desorption drying temperature set at 36℃.

[0162] Comparative Example 5:

[0163] Raw material preparation: *Eupolyphaga sinensis* (a type of insect) was selected, and the cleaning and processing were the same as in Example 5. The pieces were cut into 5cm pieces. The volatile alkaline substances test was satisfactory.

[0164] Low-temperature freezing storage: The freezing temperature is set to -12℃ for 6 days.

[0165] Homogenization: Add cold distilled water to the ground beetle homogenate at a ratio of 1:1.5 and homogenize for 15 minutes.

[0166] Freeze-thaw extraction: The freezing temperature was set to -15℃ for 10 hours, and the thawing temperature was set to 40℃ for 10 hours. The freeze-thaw process was repeated only once.

[0167] Separation steps

[0168] Low-speed centrifugation: 5000 rpm for 7 minutes.

[0169] High-speed centrifugation: 8500 rpm for 7 minutes.

[0170] Ultrafiltration separation: A filter membrane with a molecular weight cutoff of 250,000 is used, the filtration flow rate is 4L / min, and the temperature is 23℃.

[0171] Freeze-drying: Pre-freezing temperature -20℃, time 2 hours. Sublimation temperature 0℃, sublimation time 7 hours, and desorption drying temperature set at 40℃.

[0172] Experiment 1: Protein Content Detection Experiment

[0173] Experimental procedure and sample preparation: Gecko extract was prepared according to the process of Example 1 and Comparative Example 1, and 10 mL of each extract sample was taken.

[0174] The samples were stored at 4°C to prevent protein degradation.

[0175] Preparation of standard solutions: Standard solutions were prepared using bovine serum albumin (BSA) with concentrations ranging from 0, 50, 100, 200, 300, 400, to 500 μg / mL.

[0176] Color reaction:

[0177] Take 100 μL of the standard solution and sample solution respectively, and add them to a 96-well plate.

[0178] Add 100 μL of Coomassie Brilliant Blue dye to each well and react at room temperature for 5 minutes.

[0179] Absorbance measurement:

[0180] The absorbance was measured using a spectrophotometer (595 nm), and each sample was measured three times.

[0181] Calculate the protein concentration (μg / mL) in the sample based on the standard curve.

[0182] Data processing: Calculate the total protein content based on the sample volume and record the results.

[0183] Table 1. Protein content detection results of the examples and comparative examples.

[0184] Sample number Absorbance (OD) Protein concentration (μg / mL) Total protein content (mg) Example 1-1 0.341 215.6 2.16 Examples 1-2 0.367 227.4 2.27 Examples 1-3 0.359 223.1 2.23 Comparative Example 1-1 0.187 113.8 1.14 Comparative Examples 1-2 0.194 116.4 1.16 Comparative Examples 1-3 0.183 112.1 1.12

[0185] Experimental data show that the process in this invention significantly improves the protein content of the extract. In the examples, more freeze-thaw extractions were performed, the freezing temperature was lower, and the fractionation accuracy of centrifugation and ultrafiltration was higher. These optimized conditions combined resulted in more complete release of intracellular proteins, ultimately leading to a significant improvement in extraction efficiency. In the comparative example, insufficient freeze-thaw conditions resulted in low cell disruption efficiency and limited protein release. These differences directly demonstrate the decisive influence of process design on the results.

[0186] During freeze-thaw cycles, the low-temperature ice crystal effect is a key factor. In the example, multiple freeze-thaw cycles repeatedly subjected the cell membrane to mechanical damage from ice crystals, leading to a gradual accumulation of protein extravasation. In contrast, the comparative example only underwent a single freeze-thaw cycle, and the ice crystal effect was not fully realized. The setting of the freezing and thawing temperatures is also crucial. The temperature range chosen in the example precisely matched the protein's stability window, avoiding secondary damage to the active ingredients. The high thawing temperature in the comparative example clearly exacerbated protein degradation and denaturation; this crude treatment inevitably prevented efficient extraction.

[0187] Furthermore, the examples demonstrate how molecular weight fractionation during centrifugation and ultrafiltration further optimized the quality of the extract. The extraction of small molecule proteins depends not only on cell disruption techniques but also on subsequent separation processes. In the comparative examples, both the centrifugation speed and the choice of ultrafiltration membrane deviated from their optimal ranges, resulting in decreased protein purity and concentration. These subtle deviations clearly reveal the ingenuity and technical value of the process design in this invention.

[0188] Experiment 2: Small Molecule Ratio Analysis Experiment

[0189] Sample preparation:

[0190] Earthworm extracts were prepared according to the processes of Example 2 and Comparative Example 2, respectively.

[0191] Take 10 mL of supernatant from each sample group, dilute it twice, and use it for later use.

[0192] Detection method:

[0193] The molecular weight distribution of the samples was analyzed using high performance liquid chromatography-gel permeation chromatography (HPLC-GPC).

[0194] Chromatographic column: TSKgel G2000SWXL.

[0195] Mobile phase: 0.1M phosphate buffer (pH 7.0), flow rate 1.0 mL / min.

[0196] Detector: Ultraviolet detector, wavelength set at 220nm.

[0197] Injection volume: 20 μL.

[0198] Operating steps:

[0199] Inject the diluted samples into the HPLC system and run the program.

[0200] Record the peak area of ​​components with a molecular weight range <10kDa and calculate their proportion of the total peak area (small molecule percentage %).

[0201] The standard curve was constructed from peptide solutions of known molecular weights (range: 2kDa-50kDa) for molecular weight distribution analysis.

[0202] Data processing:

[0203] Each sample was measured twice, and the average value was taken. The entire experiment was conducted at room temperature, and no preservatives were used on the samples.

[0204] Table 2. Results of small molecule ratio detection in the examples and comparative examples.

[0205]

[0206]

[0207] The data shows that the proportion of small molecules in the extract of the example is close to 98%, significantly higher than that of the comparative example. This is the result of the combined effect of freeze-thaw extraction and fractionation. In freeze-thaw extraction, the expansion effect of ice crystals directly disrupts the cell membrane structure, allowing more small molecules to seep out. Repeated freeze-thaw operations not only increase the release amount but also provide more efficient preconditions for subsequent separation. In the comparative example, only one freeze-thaw cycle resulted in limited cell wall disruption, naturally restricting the release of small molecules.

[0208] The precision of the fractionation separation technology is another key factor. The centrifugation and ultrafiltration parameters in the examples were rigorously optimized, particularly the selection of the molecular weight cutoff, to ensure the enrichment of small-molecule active ingredients. In the comparative example, however, the ultrafiltration membrane had lower separation precision, resulting in the inclusion of high-molecular-weight impurities in the extract, thus reducing the proportion of small molecules. Based on the HPLC data, it can be inferred that inadequate flow rate and temperature control in the comparative example may also have affected the separation efficiency of small-molecule components.

[0209] Furthermore, the setting of the melting temperature is also noteworthy. The low-temperature melting conditions used in the examples avoided the degradation of small molecule peptides, while the high-temperature melting in the comparative examples easily caused denaturation or aggregation of some small molecules. These differences collectively determine the significant difference in the proportion of small molecules in the final extract. Through this experiment, it is evident that the low-temperature freeze-thaw and high-precision separation process designed in the examples performs superiorly in the extraction and enrichment of active ingredients.

[0210] Experiment 3: Amino Acid Content Determination Experiment

[0211] Sample preparation:

[0212] Snakehead extract was prepared according to the processes of Example 3 and Comparative Example 3.

[0213] Take 5 mL of each extract and dilute it 10 times with distilled water.

[0214] It passes through a 0.45μm filter membrane to remove suspended solids and impurities.

[0215] Detection method:

[0216] Instruments and conditions: An automated amino acid analyzer (Hitachi L-8900) was used.

[0217] Chromatographic column: Amino acid-specific column (separation range covers 20 amino acids).

[0218] Mobile phase: 0.2M citrate buffer, flow rate: 0.4 mL / min.

[0219] Temperature: Column temperature set to 50℃.

[0220] Sample injection: Inject 20 μL of sample each time, and the analysis time is 60 minutes.

[0221] Standards: Amino acid standards (manufactured by Sigma) with a concentration range of 0-500 μM were used to plot a standard curve.

[0222] Experimental setup: Each sample was measured 3 times, the peak area of ​​each amino acid was recorded, and the total amino acid content and free amino acid content were calculated.

[0223] Table 3. Amino acid content detection results of the examples and comparative examples.

[0224] Sample number Total amino acid content (μg / mL) Free amino acid content (μg / mL) Example 3-1 1356.4 1085.12 Example 3-2 1329.8 1090.436 Example 3-3 1340.2 1045.356 Comparative Example 3-1 947.3 682.0559 Comparative Example 3-2 953.5 705.59 Comparative Example 3-3 960.1 672.07

[0225] The differences in amino acid detection results were quite striking. The total amino acid content of the example was more than 40% higher than that of the comparative example, and the proportion of free amino acids was nearly doubled. These data are not accidental. With the increase of freeze-thaw extraction times, the cell structure is repeatedly damaged by ice crystals, and the possibility of proteins and peptides breaking down into small amino acids increases significantly. In the comparative example, only one freeze-thaw cycle was performed, resulting in limited release; the amino acids inside the cells remained trapped in the incompletely ruptured cells and were difficult to release.

[0226] The setting of the melting temperature is also crucial. The low-temperature melting in the examples gently activates protease activity, causing the protein to slowly decompose into free amino acids, while avoiding enzyme inactivation caused by high-temperature melting. In the comparative example, while high-temperature melting accelerated the extraction speed, it resulted in the loss of some protease activity, limiting the release of amino acids. Higher melting temperatures may also cause amino acid degradation, further affecting the extraction efficiency.

[0227] Centrifugation and ultrafiltration also played significant roles. The high-precision centrifugation in the examples removed large molecular weight impurities, creating conditions for amino acid enrichment. The molecular weight cutoff of the ultrafiltration membrane was set within a reasonable range, allowing for the sufficient enrichment of small molecular weight amino acids. In contrast, in the comparative example, due to unreasonable separation parameters, large molecular weight impurities were introduced, diluting the amino acid concentration. Ultimately, the extraction efficiency demonstrated in the examples far exceeded that of the comparative example.

[0228] Experiment 4: Extraction Efficiency Experiment

[0229] Sample preparation:

[0230] Extracts of gecko, earthworm, and snakehead were prepared according to the processes of Examples 1, 2, 3 and Comparative Examples 1, 2, 3, respectively.

[0231] Take 10 mL of each sample, record the initial liquid mass, and store at 4°C for later use.

[0232] Extract quality determination:

[0233] Transfer 10 mL of the extract to an oven at 105 °C and dry to constant weight.

[0234] Record the dried mass of the residue using an electronic balance, retaining two decimal places.

[0235] Extraction efficiency is calculated as: extract mass (mg) / raw material mass (g) × 100%.

[0236] Experimental setup:

[0237] Each sample was measured twice, and the average value was taken.

[0238] Each experiment was conducted independently to avoid cross-contamination.

[0239] Table 4 Extraction efficiency test results

[0240] Sample number Initial mass of extract (g) Residue mass (mg) Extraction efficiency (%) Example 1-1 10.02 92.8 9.26 Examples 1-2 10.05 94.5 9.4 Example 2-1 9.96 87.2 8.75 Example 2-2 10.03 89.5 8.93 Example 3-1 10.01 96.3 9.62 Comparative Example 1-1 9.99 63.5 6.35 Comparative Examples 1-2 10.02 65.1 6.5 Comparative Example 2-1 10 70.8 7.08 Comparative Example 2-2 10.04 72.3 7.21 Comparative Example 3-1 10 68.2 6.82 Comparative Example 3-2 9.98 67.6 6.78

[0241] The data comparison of extraction efficiency is immediately apparent. In the examples, through optimized freeze-thaw and separation techniques, the extraction efficiency was generally close to or above 9%, significantly better than the 6-7% of the comparative example. This is not simply a numerical difference, but a reflection of the process design itself. The repeated freeze-thaw cycles in the examples fully unlocked the potential for releasing active ingredients after the cell membranes were mechanically damaged by ice crystals. In contrast, the comparative example only underwent a single freeze-thaw cycle, resulting in limited cell wall disruption and consequently, a lower extraction efficiency. This result was within expectations.

[0242] Besides the number of freeze-thaw cycles, the thawing conditions are also a key factor determining efficiency. The examples used lower thawing temperatures, which effectively activated enzyme activity, allowing proteins and peptides to further degrade into smaller, more easily released molecules. In contrast, the high-temperature thawing in the comparative examples was detrimental in terms of both cell penetration and the stability of active ingredients. Some components might even aggregate or degrade due to high temperatures, directly reducing the quality of the extract. Experimental data strongly confirms this.

[0243] The contribution of separation technology cannot be ignored. In the examples, the parameters of centrifugation and ultrafiltration were precisely adjusted, enabling the efficient separation of high-molecular impurities, leaving behind a purified, high-concentration extract. In contrast, the coarse separation in the comparative example failed to effectively remove large-molecular interfering components. Ultimately, this difference in separation efficiency directly affected the purity and total amount of the extract.

[0244] Experiment 5: Moisture Content Test of Final Product

[0245] Sample preparation:

[0246] Agkistrodon halys extract powder was prepared according to the processes of Example 4 and Comparative Example 4, respectively.

[0247] 2g of each product group was randomly sampled for moisture content determination.

[0248] The samples were stored in sealed bags to avoid the influence of external humidity.

[0249] Measurement method:

[0250] Instruments and setup: A Karl Fischer moisture titrator (Mettler Toledo V20) was used.

[0251] The titration solvent was anhydrous methanol, and the concentration of Karl Fischer reagent titrant was 5 mg / mL.

[0252] Operating steps:

[0253] The sample was added to a microwave digestion vessel and heated to 60°C to release moisture.

[0254] The water content released from the sample was determined by potentiometric titration with Karl Fischer reagent during the titration process.

[0255] Data Records:

[0256] Each sample was measured twice, and the average value was recorded.

[0257] Experimental setup:

[0258] A control group (comparative example) and an experimental group (example) were set up to test the effect of different processes on moisture content control.

[0259] The ambient temperature is maintained at 25℃ and the humidity is controlled below 40%.

[0260] Table 5. Results of moisture content detection in the dry powder of the examples and comparative examples.

[0261] Sample number Sample weight (g) Titration solution volume (mL) Moisture content (%) Example 4-1 2.03 0.56 5.48 Example 4-2 2.02 0.54 5.35 Comparative Example 4-1 2.01 1.12 11.15 Comparative Example 4-2 2.04 1.07 10.85

[0262] The experimental data comparison clearly shows that the moisture content of the dry powder in the example was consistently controlled below 6%, while the moisture content of the comparative example was close to 11%, significantly higher than double. This result is directly attributed to the precision of the freeze-drying process. The low-temperature pre-freezing and vacuum sublimation drying in the example effectively avoided residual moisture inside the sample through a gentle water sublimation process. In contrast, the sublimation temperature in the comparative example was too high, and the vacuum control was insufficient, resulting in incomplete moisture removal and a significantly inferior drying effect.

[0263] Moisture content is crucial for the long-term stability of a product. The low moisture content in the dry powder of the example reduces the likelihood of microbial growth and oxidation, thus extending shelf life. In contrast, the high moisture content of the comparative example not only makes the product susceptible to moisture and clumping, but also may develop off-odors or become ineffective during storage due to microbial activity. This difference fully demonstrates the profound impact of precise control in freeze-drying technology on product quality.

[0264] The temperature setting and time arrangement of the drying process directly determine the moisture content. The example uses staged pre-freezing, sublimation, and desorption drying to ensure more thorough moisture release and avoid component damage caused by high temperatures. In the comparative example, due to improper adjustment of drying parameters, the moisture release process was interrupted, and residual moisture remained inside the sample after drying.

[0265] Experiment 6: Product Stability Test

[0266] Sample preparation:

[0267] Gecko extract powder was prepared according to the processes of Example 1 and Comparative Example 1.

[0268] Take 5g of sample from each group, divide it into aluminum foil bags, seal them, and use them as test samples.

[0269] Storage conditions were set in two groups: room temperature (25℃±2℃) and high temperature (40℃±2℃).

[0270] Detection method:

[0271] Protein content detection:

[0272] The protein content of each sample was determined using the Coomassie Brilliant Blue method, following the procedure in Experiment 1, and the data changes were recorded monthly.

[0273] Detection of free amino acid content:

[0274] The content of free amino acids was determined using an automated amino acid analyzer, following the method described in Experiment 3.

[0275] Total extract quality:

[0276] Take a 2g sample every month, determine the mass of the residue after drying according to the method in Experiment 4, and calculate the total amount of extract.

[0277] Experimental setup:

[0278] Each group of samples was tested once a month for 6 consecutive months.

[0279] To minimize environmental interference, the humidity of the storage environment should be controlled below 40%.

[0280] Table 6. Product stability test results (stored at room temperature for 6 months)

[0281] Sample number month Protein content (mg / g) Free amino acid content (mg / g) Extract quality (%) Example 1-1 October 346.2 176.8 9.42 February 342.1 170.2 9.33 April 335.7 165.8 9.15 June 330.8 160.4 9.03 Comparative Example 1-1 October 218.5 105.6 6.78 February 199.2 91.2 6.35 April 178.6 79.4 6.02 June 160.3 66.7 5.74

[0282] The results are readily apparent: the decrease in protein and free amino acid content in the product of the example at room temperature was far less than that of the comparative example, and the quality of the extract remained stable. In the example, thanks to the precise control of freeze-drying, the dry powder had low moisture content, and the oxidative degradation reaction was almost completely suppressed. In contrast, the high-moisture product of the comparative example was more prone to moisture absorption during storage, resulting in accelerated protein oxidation, significant decomposition of free amino acids, and a much faster rate of quality deterioration. These changes vividly demonstrate the decisive role of different drying processes in the stability of the final product.

[0283] The unique advantages of freeze-drying technology lie not only in its efficient dehydration but also in the protection of the active ingredient structure by the gentle sublimation process. The process conditions in the example were designed precisely, avoiding the damage to proteins and amino acids caused by high temperatures while minimizing residual moisture in the product. In contrast, the comparative example showed that due to excessively high sublimation temperatures, some proteins had denatured, resulting in a significant decrease in stability. This unreasonable process setting directly led to quality loss during storage.

[0284] Oxidation reactions are particularly active under high humidity conditions. The decrease in free amino acid content in the comparative example was especially significant, which is closely related to the high moisture content. The dried product of the examples exhibited extremely high stability due to its low moisture content, and its antioxidant capacity was clearly far superior to that of the comparative example.

[0285] Experiment 7: Extraction Experiment of Pharmacologically Related Components

[0286] Sample preparation:

[0287] Earthworm extracts were prepared according to the processes of Example 5 and Comparative Example 5, respectively.

[0288] Take 10 mL of each extract, dilute it to 50 mL with cold distilled water, filter it, and set it aside.

[0289] Detection method:

[0290] Analysis using liquid chromatography-mass spectrometry (LC-MS / MS):

[0291] Chromatographic column: C18 reversed-phase column (150×4.6mm, 5μm particle size).

[0292] Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution.

[0293] Gradient elution: Initially 95% A, 5% B, linear gradient to 50% A, 50% B over 5 minutes, hold for 5 minutes.

[0294] Flow rate: 0.3 mL / min; Column temperature: 30℃.

[0295] Target ingredient:

[0296] Small molecule proteins: Detection of target molecules with molecular weights ranging from 1kDa to 5kDa using characteristic ion pairs.

[0297] Free amino acids: The total free amino acid concentration was quantitatively analyzed by comparing with the standard curve.

[0298] Injection volume:

[0299] Each injection was 20 μL, and the analysis was performed continuously, with each sample group measured twice.

[0300] Experimental setup:

[0301] Maintain room temperature (25℃) to avoid secondary degradation of the sample due to high temperature.

[0302] The data is automatically collected by the instrument, and the peak area and target component concentration are analyzed using standardized software.

[0303] Table 7. Detection results of pharmacodynamic-related components in the examples and comparative examples.

[0304]

[0305]

[0306] Experiments showed that the concentrations of small molecule proteins and free amino acids extracted in the examples were significantly higher than those in the comparative example, and this difference was by no means accidental. The freeze-thaw extraction process in the examples, through repeated freeze-thaw cycles, resulted in extremely thorough mechanical disruption of the cell membrane by ice crystals, allowing for the smooth release of small molecule proteins from the cells. In contrast, the comparative example only underwent one freeze-thaw cycle, significantly reducing the release efficiency; many active ingredients remained trapped within the unruptured cells, directly limiting the effectiveness of the extraction.

[0307] Setting the appropriate melting temperature is also crucial. The low-temperature melting conditions in the example not only avoided secondary damage to small-molecule proteins caused by high temperatures but also preserved enzyme activity, allowing for the full release of active ingredients. In contrast, the higher melting temperature in the comparative example resulted in a significant decrease in enzyme activity, and some small-molecule proteins may even have aggregated or denatured due to overheating. This difference in process details resulted in lower concentrations of small-molecule proteins and free amino acids in the extract of the comparative example compared to the example.

[0308] Furthermore, the enrichment effect of separation technology on target components is significant. The examples employed precise centrifugation and ultrafiltration to effectively remove high-molecular-weight impurities, allowing for further concentration of small-molecule proteins. In contrast, the comparative example showed a less refined separation process, resulting in more residual large-molecular-weight impurities and diluting the concentration of active ingredients.

[0309] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for the preparation of fresh animal medicine, characterized in that, The method comprises the following steps: Step (1) cleaning treatment: removing non-medicinal parts of fresh animal medicine, cutting and cleaning; Step (2) low-temperature frozen storage: freezing the cleaned fresh animal medicine at-15℃ to-25℃ environment; Step (3) homogenate treatment: cutting the frozen fresh animal medicine and adding equal amount of water to prepare homogenate under the condition that the ambient temperature is not higher than 19℃; Step (4) freeze-thaw extraction: freezing the homogenized material at-25℃±2℃ for 20-24 hours, then thawing at 37℃±1℃ for 20-24 hours, repeating 2-4 times; Step (5) fractionation: sequentially performing low-speed centrifugation, high-speed centrifugation and ultrafiltration separation on the freeze-thawed material; The low-speed centrifugation in step (5) comprises the following conditions: The centrifuge speed is 3000-7000 rpm, and the time is 10-20 minutes; The centrifugation is performed 3 times, and the liquid flow rates are controlled to be 15 L / min, 12 L / min and 10 L / min, respectively; The high-speed centrifugation in step (5) comprises the following conditions: The high-speed centrifuge speed is 8000-12000 rpm, and the time is 5-10 minutes; The liquid flow rate is 5 L / min or less; The ultrafiltration separation in step (5) comprises: Using an ultrafiltration membrane with a molecular weight cut-off of 50,000 or less; The filtration flow rate is 1-3 L / min, and the filtration temperature is controlled to be below 19℃; Step (6) freeze-drying: freeze-drying the separated liquid, wherein the pre-freezing temperature is-35℃ or lower, the sublimation temperature is-20℃ to-5℃, and the drying temperature is 35℃±2℃.

2. A process for the preparation of fresh animal medicine as claimed in claim 1, wherein, The homogenate treatment in step (3) comprises: Adding distilled water or sterile water in a ratio of 1:1 during homogenization; The homogenization time is 20-40 minutes.

3. A process for preparing fresh animal medicine as claimed in claim 1, wherein, The freeze-thaw extraction in step (4) comprises: During the freeze-thaw process, the material is frozen for 20-24 hours under the freezing condition; In the thawing stage, the material is thawed in a water bath environment for 20-24 hours, and the freeze-thaw cycle number is 3.

4. A process for preparing fresh animal medicine as claimed in claim 1, wherein, The freeze-drying in step (6) comprises the following conditions: The pre-freezing time is 1-3 hours, and the pre-freezing temperature is-35℃ or lower; The sublimation drying time is 6-12 hours, and the vacuum degree is 35 Pa or less; The water content of the final product during the drying process is ≤6%.

5. A process for preparing fresh animal medicine as claimed in claim 1, wherein, The content of volatile basic substances in the fresh animal medicine in the cleaning step is not more than 33 mg / 100 g calculated by nitrogen.

6. A process for preparing fresh animal medicine as claimed in claim 1, wherein, The treatments in steps (3) to (6) are all performed under the condition that the temperature is not higher than 19℃.

Images