Preparation process of fresh animal medicine

Abstract

The invention relates to the technical field of animal medicine preparation, and discloses a fresh animal medicine preparation process which comprises the following steps: (1) cleaning treatment: removing non-medicinal parts of a fresh animal medicine, cutting into blocks and cleaning; (2) low-temperature freezing storage: putting the cleaned fresh animal medicine in an environment of-15 DEG C to-25 DEG C for freezing storage; (3) homogenizing treatment: adding the same amount of water into the frozen fresh animal medicine dices under the condition that the environment temperature is not higher than 19 DEG C to prepare homogenate; and (4) freeze thawing extraction. By adopting the technical scheme of low-temperature freezing storage and repeated freezing and thawing extraction, physical damage of cell membranes is realized through formation and dissolution of ice crystals, so that active ingredients such as proteins and enzymes in fresh animal medicines are fully released, protein denaturation and activity loss caused by heating are avoided, the extraction rate of effective ingredients is remarkably increased, and the production cost is reduced. The defect that natural activity of fresh animal medicine is difficult to preserve in a traditional method is overcome.

Description

Technical Field

[0001] The invention relates to the technical field of animal medicine preparation, in particular to a process for preparing fresh animal medicine. Background Art

[0002] Traditional Chinese medicine occupies an important position in traditional medicine with its unique curative effect and rich resources. Animal medicine is considered to be "sentient flesh and blood" because of its similarity to human structure and unique biologically active ingredients. It plays an irreplaceable role in the treatment of acute and severe diseases and complex internal medicine diseases. Animal medicine is rich in active substances such as proteins, enzymes and peptides, which directly affect the efficacy of the medicine. However, fresh animal medicine is very easy to spoil due to its high protein and high water content. Processing and preservation have always been technical difficulties in the industry.

[0003] At present, the processing and preservation of fresh animal medicines mostly adopt methods such as baking and shade drying. Although these processes can extend the storage period, they often damage the active ingredients. For example, high-temperature decoction will cause protein denaturation and polypeptide degradation, and the biological activity will be significantly reduced; during the drying process, some key ingredients may become ineffective due to oxidation. In addition, although the traditional baking and shade drying processes avoid high-temperature treatment, their water removal efficiency is low, and the product has a high residual moisture content, which can easily affect the stability of the product. Existing technologies are not yet able to efficiently extract the active ingredients of fresh animal medicines while ensuring the high purity and long-term stability of the finished product.

[0004] A major problem with the prior art is that it is difficult to balance the extraction efficiency of active ingredients and the stability of the products in the processing of fresh animal drugs. Due to insufficient cell wall disruption in the extraction process, the key ingredients are not released enough, resulting in a low content of active ingredients. At the same time, the water is not completely removed during the drying stage, making the products easy to deteriorate and lose their effectiveness during storage. Therefore, there is an urgent need for a preparation process that can efficiently extract the active ingredients of fresh animal drugs and ensure low moisture and high stability of the final product. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a preparation process for fresh animal medicine, which solves the problems of low extraction efficiency of active ingredients and poor product stability in the processing technology of fresh animal medicine in the prior art.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A process for preparing fresh animal medicine, comprising the following steps: Step (1) cleaning: removing non-medicinal parts of fresh animal medicine, cutting into pieces and cleaning; Step (2) low temperature frozen storage: the cleaned fresh animal drug is placed in a -15°C to -25°C environment for freezing and storage; Step (3) homogenization: cutting the frozen fresh animal medicine into pieces and adding an equal amount of water to prepare a homogenate at an ambient temperature not higher than 19°C; Step (4) freeze-thaw extraction: freeze the homogenized material at -25°C ± 2°C for 20-24 hours, and then thaw it at 37°C ± 1°C for 20-24 hours, repeating 2-4 times; Step (5) fractionation: the frozen and thawed material is subjected to low-speed centrifugation, high-speed centrifugation and ultrafiltration separation in sequence; Step (6) freeze drying: the separated liquid is freeze dried at low temperature, wherein the pre-freezing temperature is below -35°C, the sublimation temperature is between -20°C and -5°C, and the drying temperature is 35°C±2°C.

[0007] Preferably, the homogenization treatment in step (3) comprises: Add distilled water or sterile water in a 1:1 ratio during the homogenization process; The homogenization time is 20-40 minutes.

[0008] Preferably, the freeze-thaw extraction in step (4) comprises: During the freeze-thaw process, the material is frozen for 20-24 hours under freezing conditions; During the thawing stage, the material is thawed in a water bath for 20-24 hours, and the number of freeze-thaw cycles is 3 times.

[0009] Preferably, the low-speed centrifugation in step (5) includes the following conditions: The centrifuge speed is 3000-7000rpm, and the time is 10-20 minutes; The centrifugation was performed three times, and the flow rates of the drug solution were controlled at 15 L / min, 12 L / min, and 10 L / min, respectively.

[0010] Preferably, the high-speed centrifugation in step (5) includes the following conditions: The speed of the high-speed centrifuge is 8000-12000rpm, and the time is 5-10 minutes; The flow rate of the drug solution is below 5L / min.

[0011] Preferably, the ultrafiltration separation in step (5) comprises: Use ultrafiltration membrane with a molecular weight cutoff of less than 50,000; The filtration flow rate is 1-3 L / min, and the filtration temperature is controlled below 19°C.

[0012] Preferably, the freeze-drying in step (6) comprises the following conditions: The pre-freezing time is 1-3 hours, and the pre-freezing temperature is below -35°C; Sublimation drying time is 6-12 hours, and the vacuum degree is below 35Pa; The moisture content of the final product during the drying process is ≤6%.

[0013] Preferably, the volatile alkaline substance content of the fresh animal medicine in the washing step does not exceed 33 mg / 100 g in terms of nitrogen.

[0014] Preferably, each treatment from step (3) to step (6) is carried out in an environment at a temperature not higher than 19°C.

[0015] The present invention provides a process for preparing fresh animal medicine. It has the following beneficial effects: 1. The present invention adopts the technical scheme of low-temperature freezing storage and repeated freeze-thaw extraction to achieve physical destruction of cell membranes through the formation and dissolution of ice crystals, so that active ingredients such as proteins and enzymes in fresh animal medicines are fully released. Compared with the high-temperature decoction method in the prior art, it avoids protein denaturation and activity loss caused by heating, significantly improves the extraction rate of effective ingredients, and solves the problem that traditional methods are difficult to preserve the natural activity of fresh animal medicines.

[0016] 2. The present invention combines fractional centrifugation and ultrafiltration separation technology to extract small molecule active ingredients in a refined manner. Through multi-stage centrifugation and membrane filtration, the proportion of active ingredients with a molecular weight below 10kDa reaches more than 98%, which is much higher than the purification effect of traditional filter screens, filter cloths or filter papers. In the prior art, the extraction purity of active ingredients is low and there are many impurities, which makes it difficult to meet the needs of high-efficiency drug preparation. The present invention completely solves this problem and makes the drug quality more stable.

[0017] 3. The present invention realizes the controllability of efficient component extraction and separation through clear low-temperature freezing, freeze-thaw cycle times and temperature parameter design. The existing technology often relies on experience operation, and the process repeatability is poor, which affects the extraction efficiency and product consistency. The present invention solves the shortcomings of technical instability and difficulty in standardizing efficacy in traditional processes through systematic process flow design, and provides a new solution for the modern preparation of fresh animal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention is a flow chart of the method. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Please refer to the attached Figure 1The embodiment of the present invention provides a process for preparing fresh animal medicine, comprising: Step 1: Prepare the ingredients Cleaning and cutting: The fresh animal medicine is initially processed, including cleaning to remove impurities, cutting off non-medicinal parts, and cutting the medicinal materials into small pieces of 3-5 cm to facilitate subsequent processing.

[0021] Testing: Test the volatile alkaline content (in terms of nitrogen) in the medicinal materials to ensure that it is ≤33mg / 100g to ensure the quality of the raw materials and the freshness of the medicinal ingredients.

[0022] During the storage of fresh animal drugs, protein decomposition and corruption will generate volatile alkaline substances. This detection link can timely remove unqualified raw materials to prevent them from affecting the extraction efficiency and drug stability of subsequent processes. At the same time, the cutting operation can increase the contact area between the medicinal materials and the freezing medium and subsequent processing equipment, enhancing the freezing and extraction effects.

[0023] Step 2: Freeze and store at low temperature Store the cleaned medicinal materials in a frozen environment at -15℃ to -25℃ for no more than 30 days.

[0024] Low temperature environment can inhibit the growth and metabolic activity of microorganisms in fresh animal drugs, and effectively delay the degradation process of proteins and enzymes, thereby maintaining their natural activity. In addition, water forms ice crystals when frozen, which provides a physical basis for subsequent freeze-thaw extraction. The formation of ice crystals will cause the cell membrane and cell wall to be subjected to mechanical forces, creating favorable conditions for the release of active ingredients.

[0025] Step 3: Repeated freeze-thaw extraction and homogenization treatment: Add distilled water or sterile water to the frozen medicinal materials at an ambient temperature of ≤19°C, and make a homogenate in a 1:1 ratio. The homogenization time is 20-40 minutes.

[0026] 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.

[0027] The freeze-thaw process was repeated 2-4 times.

[0028] Destruction of cell structure: When freezing, water forms ice crystals, which expand in volume and cause the cell membrane and cell wall to rupture due to mechanical forces.

[0029] As the number of freeze-thaw cycles increases, the repeated formation and melting of ice crystals further weakens the integrity of the cell membrane, thereby releasing active ingredients such as proteins, enzymes and amino acids.

[0030] Changes in salt concentration: During the freezing process, the concentration of salt substances in the liquid increases, causing changes in cell membrane permeability and further promoting the extravasation of intracellular components.

[0031] Gentle extraction: Freeze-thaw extraction completely avoids the destruction of active ingredients by high-temperature decoction, and helps maintain the natural structure and functional activity of proteins and enzymes.

[0032] Step 4: Fractionation and low-speed centrifugation: The frozen-thawed material is centrifuged three times at low speed using a plate centrifuge, with the speed controlled at 3000-7000 rpm and the time being 10-20 minutes.

[0033] The centrifugal flow rate was controlled at 15 L / min (first time), 12 L / min (second time), and 10 L / min (third time).

[0034] High-speed centrifugation: The supernatant after low-speed centrifugation is placed in a tube centrifuge and centrifuged twice at high speed: The first rotation speed is 8000-10000rpm, the flow rate is ≤5L / min, and the time is 5-10 minutes; The second rotation speed is 10000-12000rpm, the flow rate is ≤2.5L / min, and the time is 5-10 minutes.

[0035] Ultrafiltration separation: The liquid after high-speed centrifugation is filtered through an ultrafiltration membrane with a molecular weight cutoff of less than 50,000, and the filtration temperature is controlled to be ≤19°C and the filtration flow rate is 1-3L / min.

[0036] Low-speed centrifugation gradually removes coarse particles and impurities in the drug solution; High-speed centrifugation further separates small particle impurities and protein aggregates.

[0037] Molecular screening: Ultrafiltration membranes use screening mechanisms to retain only small molecule components with a molecular weight of less than 50,000, including small molecule proteins, peptides and amino acids, which have better pharmacological activity and bioavailability.

[0038] Avoid heat damage: The whole process is carried out in a low temperature environment to ensure the stability of the extract.

[0039] Step 5: Low-temperature freeze drying pre-freezing stage: Place the separated drug solution in a freeze dryer with a pre-freezing temperature of ≤-35°C for 1-3 hours.

[0040] Sublimation drying: Under the condition of vacuum degree <35Pa, the temperature is controlled at -20℃ to -5℃ for sublimation drying for 6-12 hours.

[0041] Analytical drying: The temperature is gradually raised to 35°C ± 2°C for 6 hours until the water content of the medicinal materials is ≤ 6%.

[0042] Drying mechanism: Freeze drying directly converts the water in the drug solution from solid to gas through sublimation, avoiding protein denaturation and degradation of active substances that may be caused by high-temperature drying.

[0043] Enhanced stability: Reducing the moisture content to below 6% helps improve the stability of the product and extend its shelf life.

[0044] Integrity protection: Under low-temperature drying conditions, the proteins, enzymes and small molecule components in fresh animal medicines can maintain their natural structure, thereby retaining the biological activity of the medicinal materials.

[0045] This process overcomes the loss of active ingredients caused by high temperature treatment in traditional processes by using low temperature freezing, repeated freeze-thaw extraction and fractionation separation technology, combined with the application of low temperature freeze drying. In each technical link, reasonable design is carried out based on scientific principles such as cell structure, physical separation and molecular stability, achieving efficient extraction and preservation of active ingredients of fresh animal drugs.

[0046] Example 1: Preparation of fresh animal medicine of Gecko Step 1: Prepare the ingredients Take fresh geckos, remove the non-medicinal parts, and rinse thoroughly with cold water. Cut into blocks of about 4cm×4cm square, weigh with an electronic scale, and process 2kg of raw materials per batch. Test the content of volatile alkaline substances, which is 28mg / 100g in terms of nitrogen, which meets the requirements.

[0047] Step 2: Freeze and store at low temperature Place the cut geckos in a low-temperature freezer at -20°C. Set the storage time to 10 days. During this period, check the ice coverage to ensure uniform freezing.

[0048] Step 3: Homogenization Put the frozen gecko block into the bone mud grinder, add cold distilled water in a 1:1 ratio, set the grinding speed to 800rpm, and the homogenization time to 30 minutes. The homogenized material is uniform and has no obvious particles.

[0049] Step 4: Freeze-thaw extraction Place the homogenate in a freezer, set the temperature to -25°C, and freeze for 24 hours. After taking it out, place it in a water bath at 37°C ± 1°C to thaw, and set the thawing time to 22 hours. Repeat the freeze-thaw cycle 3 times until the homogenate is significantly diluted and the detected protein concentration is significantly increased.

[0050] Step 5: Low and high speed centrifugation The frozen and thawed liquid sample was placed in a flat plate centrifuge, and the centrifugal speed was set to 5000 rpm for 15 minutes. After the coarse particles were separated, the supernatant was taken. Then it was transferred to a tubular centrifuge and centrifuged at high speed at 10000 rpm for 8 minutes to separate the tiny impurity particles.

[0051] Step 6: Ultrafiltration Separation The liquid after high-speed centrifugation was passed through an ultrafiltration membrane with a molecular weight cutoff of 50,000, the filtration temperature was maintained at 18°C, and the filtration flow rate was 2.5L / min. The extract was clear and transparent, and the proportion of small molecule proteins was significantly increased.

[0052] Step 7: Freeze Drying The ultrafiltered extract was transferred to a freeze dryer, and the pre-freezing temperature was set to -35°C for 2 hours. Sublimation drying was then performed under a vacuum degree of 30 Pa, and the sublimation temperature was controlled at -10°C for 10 hours. The final analytical drying temperature was set to 35°C, and the temperature was kept for 5 hours, and the moisture content of the obtained dry powder was 5.8%.

[0053] Embodiment 2: preparation of fresh earthworm animal medicine Step 1: Prepare the ingredients Select fresh earthworms and wash away the surface sand and impurities. Use a peeling knife to clean the surface residues and cut into sections of about 5 cm in length. Preliminary testing shows that the content of volatile alkaline substances is 30 mg / 100 g.

[0054] Step 2: Freeze and store at low temperature Place the earthworm medicinal materials in batches into the freezing equipment, set the freezing temperature to -18°C for 14 days.

[0055] Step 3: Homogenization After the frozen earthworms were taken out, they were processed by a high-speed homogenizer, and an equal amount of cold distilled water was added, and the homogenization time was set to 25 minutes. Finally, the homogenized material was in a uniform paste state without suspended solid particles.

[0056] Step 4: Freeze-thaw extraction The homogenate was transferred to a freezer, and the freezing temperature was controlled at -23°C for 20 hours. Then it was taken out and thawed in a 37°C water bath for 20 hours. After 4 freeze-thaw cycles, the protein content detection value reached a peak value.

[0057] Step 5: Centrifugation The extract was transferred to a plate centrifuge for low-speed centrifugation at 4000 rpm for 12 minutes. The supernatant was centrifuged again at 6000 rpm for 10 minutes. It was then transferred to a high-speed centrifuge at 11000 rpm for 6 minutes to completely remove the polymer impurity particles.

[0058] Step 6: Membrane Filtration The supernatant is passed through an ultrafiltration device, using a filter membrane with a molecular weight cutoff of less than 50,000, with a filtration flow rate of 3L / min and a temperature maintained at 17°C. The liquid after filtration is clear, and the main components are small molecule proteins and amino acids.

[0059] Step 7: Freeze Drying The extract was transferred to the freeze-drying equipment, with a pre-freezing temperature of -30°C for 1.5 hours. During sublimation drying, the vacuum degree was 28 Pa, the sublimation temperature was set at -15°C, and the time was 8 hours. After drying, the water content of the product was 5.2%.

[0060] Example 3: Preparation of fresh animal medicine of Snakehead moniliformis Step 1: Prepare the ingredients The internal organs and head of fresh golden snake were removed, the residue was cleaned and cut into 3 cm long segments. The volatile alkaline substance content was detected to be 32 mg / 100 g.

[0061] Step 2: Freeze and store at low temperature The freezing temperature of medicinal materials is set at -25℃ and the storage time is 7 days.

[0062] Step 3: Homogenization Take frozen golden snake, add cold distilled water at a ratio of 1:0.8 and homogenize for 35 minutes. The resulting homogenate is fine and light pink.

[0063] Step 4: Freeze-thaw extraction The homogenate was frozen at -24°C for 24 hours and then thawed in a 36°C water bath for 21 hours. The freezing and thawing cycle was repeated 3 times.

[0064] Step 5: Low speed centrifugation The frozen and thawed liquid was placed in a low-speed centrifuge at 4500 rpm for 12 minutes, and the supernatant was collected.

[0065] Step 6: High-speed centrifugation and ultrafiltration The high-speed centrifuge speed is 10,000 rpm for 7 minutes. The supernatant is passed through an ultrafiltration device to retain small molecules with a molecular weight of less than 50,000. The filtration temperature is 16°C.

[0066] Step 7: Freeze Drying The pre-freezing temperature was -33°C for 2 hours. The sublimation temperature was controlled at -12°C for 9 hours, and the final analytical drying temperature was 33°C. The moisture content of the obtained dry powder was 5.5%.

[0067] Embodiment 4: preparation of fresh Agkistrodon acutus animal medicine Steps 1-7 (same as above technical steps, adjust the number of freeze-thaw cycles to 4 times, and the frozen storage time is 12 days).

[0068] Example 5: Preparation of fresh animal medicine of Eupolyphaga sinensis Step 1-7 (same as the above technical steps, adjust the high-speed centrifugation speed to 12000 rpm and the ultrafiltration separation temperature to 15°C).

[0069] Comparative Example 1: Raw material preparation: Select fresh Gecko medicinal materials, remove non-medicinal parts and rinse, and cut into pieces of about 4 cm×4 cm. The volatile alkaline substance content test result is 28 mg / 100 g. The preparation steps are consistent with Example 1.

[0070] Low temperature frozen storage: The freezer temperature is set to -10°C and the freezing time is 15 days.

[0071] Homogenization: Add distilled water to the frozen gecko blocks at a ratio of 1:0.5, use a low-speed bone mud grinder, and homogenize for 15 minutes.

[0072] Freeze-thaw extraction: The homogenized material was frozen at -15°C for 12 hours and then thawed in a 45°C water bath for 10 hours. The freeze-thaw cycle was performed only once.

[0073] Separation steps: Low-speed centrifugation: The speed is set to 2000 rpm and the time is 10 minutes.

[0074] High-speed centrifugation: speed is 6000 rpm, time is 5 minutes.

[0075] Ultrafiltration separation: Use a filter membrane with a molecular weight cutoff of 100,000, the filtration temperature is about 25°C, and the flow rate is 5L / min.

[0076] Freeze drying: pre-freezing temperature was set at -20°C, sublimation temperature at -5°C, sublimation time at 6 hours, and analytical drying temperature at 40°C.

[0077] Comparative Example 2: Raw material preparation: Take fresh earthworm medicinal materials, process them in the same way as in Example 2, and cut them into pieces with a length of 5 cm. The volatile alkaline substance test is qualified.

[0078] Low temperature frozen storage: The freezing equipment temperature is set to -5°C and the freezing time is 5 days.

[0079] Homogenization: The frozen earthworms were processed with a high-speed homogenizer, cold water was added at a ratio of 1:1.5, and the homogenization time was shortened to 10 minutes.

[0080] Freeze-thaw extraction: The homogenate was frozen at -10°C for 6 hours and thawed in a 50°C water bath for 8 hours. The freeze-thaw process was not cycled and was performed only once.

[0081] Separation steps Low-speed centrifugation: 3000 rpm, 8 minutes.

[0082] High-speed centrifugation: speed 8000rpm, time 5 minutes.

[0083] 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°C.

[0084] Freeze drying: pre-freezing temperature -15°C, time 1 hour. Sublimation temperature 0°C, sublimation time 5 hours, analytical drying temperature set to 38°C.

[0085] Comparative Example 3: Raw material preparation: Snakehead coin The internal organs and head of fresh snakehead coin were removed and the segmentation method was the same as in Example 3. The segment length was about 3 cm. The volatile alkaline substance content met the requirements.

[0086] Low temperature frozen storage: The freezing equipment temperature is set to 0°C and the freezing time is 3 days.

[0087] Homogenization: Add cold water to the homogenate at a ratio of 1:2, and process it with a low-speed homogenizer. The homogenization time is shortened to 5 minutes.

[0088] Freeze-thaw extraction: Freezing temperature -5°C, freezing time 4 hours. Thawing condition is 4 hours in a 55°C water bath. The freeze-thaw process is not cycled and is only operated once.

[0089] Separation steps: Low-speed centrifugation: 3500 rpm, 5 minutes.

[0090] High-speed centrifugation: speed 7000rpm, time 5 minutes.

[0091] Ultrafiltration separation: Use an ultrafiltration membrane with a molecular weight cutoff of 300,000, the filtration temperature is 25°C, and the flow rate is 6 L / min.

[0092] Freeze drying: pre-freezing temperature -10°C, time 0.5 hours, sublimation temperature 5°C, sublimation time 4 hours, analytical drying temperature set to 42°C.

[0093] Comparative Example 4: Raw material preparation: The processing method of Agkistrodon acutus was the same as that in Example 4, the internal organs were removed, the head was directly cut into sections, and the length of the sections was 5 cm. The volatile alkaline substance test was qualified.

[0094] Low temperature frozen storage: The freezing temperature is set to -8°C and the freezing time is 8 days.

[0095] Homogenization: Add cold distilled water to the frozen Agkistrodon acutus homogenate at a ratio of 1:0.8, and the homogenization time is 20 minutes.

[0096] Freeze-thaw extraction: Freezing temperature is -12°C, freezing time is 8 hours, thawing temperature is 48°C, thawing time is 12 hours. Freeze-thaw cycle is only 2 times.

[0097] Separation steps Low-speed centrifugation: speed 4000 rpm, time 8 minutes.

[0098] High-speed centrifugation: speed 9000 rpm, time 6 minutes.

[0099] Ultrafiltration separation: Use an ultrafiltration membrane with a molecular weight cutoff of 150,000, the filtration temperature is 20°C, and the flow rate is 5 L / min.

[0100] Freeze drying: pre-freezing temperature -25°C, time 1 hour. Sublimation temperature -10°C, sublimation time 8 hours, analytical drying temperature set to 36°C.

[0101] Comparative Example 5: Raw material preparation: Eupolyphaga sinensis medicinal material was selected, and the cleaning process was the same as in Example 5, and the size of the cut pieces was 5 cm. The volatile alkaline substance test was qualified.

[0102] Low temperature storage: The freezing temperature is set at -12°C for 6 days.

[0103] Homogenization treatment: Add cold distilled water to the homogenate of Eupolyphaga sinensis at a ratio of 1:1.5, and the homogenization time is 15 minutes.

[0104] Freeze-thaw extraction: The freezing temperature was set at -15°C, the freezing time was 10 hours, and the thawing temperature was 40°C, the thawing time was 10 hours. Freeze-thaw was performed only once.

[0105] Separation steps Low-speed centrifugation: 5000 rpm, 7 minutes.

[0106] High-speed centrifugation: speed 8500rpm, time 7 minutes.

[0107] Ultrafiltration separation: Use a filter membrane with a molecular weight cutoff of 250,000, a filtration flow rate of 4 L / min, and a temperature of 23°C.

[0108] Freeze drying: pre-freezing temperature -20°C, time 2 hours. Sublimation temperature 0°C, sublimation time 7 hours, analytical drying temperature set to 40°C.

[0109] Experiment 1: Protein content detection experiment Experimental steps Sample preparation: Prepare the gecko extracts according to the processes of Example 1 and Comparative Example 1, and take 10 mL of each extract sample.

[0110] The samples were stored at 4°C to avoid protein degradation.

[0111] Standard solution preparation: bovine serum albumin (BSA) was used to prepare standard solutions with concentrations ranging from 0, 50, 100, 200, 300, 400, and 500 μg / mL.

[0112] Color reaction: Take 100 μL of the standard solution and sample solution respectively and add them into a 96-well plate.

[0113] 100 μL of Coomassie Brilliant Blue dye was added to each well and reacted at room temperature for 5 minutes.

[0114] Absorbance measurement: The absorbance was measured using a spectrophotometer (595 nm), and each sample was measured three times.

[0115] The protein concentration (μg / mL) in the samples was calculated based on the standard curve.

[0116] Data processing: Combine the sample volume, calculate the total protein content, and record the results.

[0117] Table 1 Protein content test results of examples and comparative examples Sample No. Absorbance (OD) Protein concentration (μg / mL) Total protein content (mg) Example 1-1 0.341 215.6 2.16 Example 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 Example 1-2 0.194 116.4 1.16 Comparative Examples 1-3 0.183 112.1 1.12 Experimental data show that the process of the present invention significantly improves the protein content of the extract. In the embodiment, the number of freeze-thaw extractions is more, the freezing temperature is lower, and the classification accuracy of centrifugation and ultrafiltration is higher. These optimized conditions are combined to make the release of intracellular proteins more sufficient, and the final extraction efficiency is significantly improved. In the comparative example, due to insufficient freeze-thaw conditions, the cell wall breaking efficiency is low and the protein release amount is limited. These differences directly reflect the decisive influence of process design on the results.

[0118] During the freeze-thaw process, the low-temperature ice crystal effect is a key factor. In the embodiment, the cell membrane is repeatedly mechanically damaged by ice crystals through multiple freeze-thaw cycles, and the amount of protein exudation gradually accumulates. However, the comparative example only undergoes freeze-thaw once, and the ice crystal effect has not yet fully taken effect. The setting of the freezing temperature and the melting temperature is also crucial. The temperature range selected in the embodiment accurately matches the stability window of the protein, avoiding secondary damage to the active ingredient. The high melting temperature in the comparative example obviously exacerbates the degradation and denaturation of the protein, and this rough treatment is doomed to fail to achieve efficient extraction.

[0119] In addition, the embodiment further optimizes the quality of the extract by controlling the molecular weight during centrifugation and ultrafiltration. The extraction of small molecule proteins not only depends on the cell wall breaking technology, but is also closely related to the subsequent separation process. In the comparative example, the centrifugal speed and the selection of ultrafiltration membrane deviated from the optimal range, resulting in a decrease in protein purity and concentration. These deviations in details clearly reveal the creativity and technical value of the process design of the present invention.

[0120] Experiment 2: Small molecule ratio analysis experiment Sample preparation: According to the processes of Example 2 and Comparative Example 2, respectively, an earthworm extract was prepared.

[0121] Take 10 mL of supernatant from each group of samples and dilute it 2 times for later use.

[0122] Detection method: The molecular weight distribution of the samples was analyzed using high performance liquid chromatography-gel permeation chromatography (HPLC-GPC).

[0123] Chromatographic column:TSKgelG2000SWXL.

[0124] Mobile phase: 0.1 M phosphate buffer (pH 7.0), flow rate 1.0 mL / min.

[0125] Detector: UV detector, wavelength set to 220nm.

[0126] Injection volume: 20 μL.

[0127] Steps: Inject the diluted samples into the HPLC system respectively and run the program.

[0128] The chromatographic peak area of ​​the component with a molecular weight range of <10 kDa was recorded, and its proportion in the total peak area (small molecule proportion %) was calculated.

[0129] A standard curve was constructed from peptide solutions of known molecular weight (range: 2 kDa-50 kDa) for molecular weight distribution analysis.

[0130] Data processing: Each sample was measured twice and the average value was taken. The whole experiment was carried out at room temperature and no preservatives were used in the samples.

[0131] Table 2 Detection results of small molecule ratios in Examples and Comparative Examples It can be seen from the data that the proportion of small molecules in the extract of the embodiment is close to 98%, which is significantly higher than that of the comparative example. This is the result of the combined effect of freeze-thaw and fractionation separation processes. In freeze-thaw extraction, the expansion effect of ice crystals directly destroys the cell membrane structure, causing more small molecules to seep out. Repeated freeze-thaw operations not only increase the release amount, but also provide a more efficient prerequisite for subsequent separation. In the comparative example, there was only one freeze-thaw, the cell wall was limited, and the released small molecules were naturally restricted.

[0132] The accuracy of fractionation separation technology is another key. The centrifugation and ultrafiltration parameters in the embodiment are strictly optimized, especially the selection of molecular weight cut-off, to ensure the enrichment of small molecule effective components. In the comparative example, the separation accuracy of the ultrafiltration membrane is low, resulting in the doping of high molecular impurities in the extract, and the proportion of small molecules is therefore reduced. Combined with HPLC data, it can be inferred that the flow rate and temperature control in the comparative example are not in place, which may also affect the separation efficiency of small molecule components.

[0133] In addition, the setting of the melting temperature is also worth noting. The low-temperature melting conditions adopted in the embodiment avoid the degradation of small molecule polypeptides, while the high-temperature melting in the comparative example easily causes denaturation or aggregation of some small molecules. These differences jointly determine the significant difference in the proportion of small molecules in the final extract. Through this experiment, it is obvious that the low-temperature freeze-thaw and high-precision separation process designed in the embodiment performs superiorly in extracting and enriching active ingredients.

[0134] Experiment 3: Amino acid content determination experiment Sample preparation: The extracts of Agkistrodon acutus were prepared according to the processes of Example 3 and Comparative Example 3.

[0135] Take 5 mL of each extract and dilute it 10 times with distilled water.

[0136] Pass through a 0.45μm filter membrane to filter out suspended matter and impurities.

[0137] Detection method: Instruments and conditions: An amino acid automatic analyzer (Hitachi L-8900) was used.

[0138] Chromatographic column: Amino acid-specific column (separation range covers 20 kinds of amino acids).

[0139] Mobile phase: 0.2 M citrate buffer, flow rate 0.4 mL / min.

[0140] Temperature: The column temperature was set at 50°C.

[0141] Sample injection: 20 μL of sample was injected each time, and the analysis time was 60 min.

[0142] Standards: Amino acid standards (manufactured by Sigma) were used with a concentration range of 0-500 μM to draw a standard curve.

[0143] 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.

[0144] Table 3: Detection results of amino acid content in examples and comparative examples Sample No. 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 The difference presented in the amino acid test results is quite clear. The total amino acid content of the embodiment is more than 40% higher than that of the control example, and the proportion of free amino acids is nearly doubled. These data are not accidental. When the number of freeze-thaw extractions increases, the cell structure is repeatedly damaged by the action of ice crystals, and the possibility of protein and polypeptide cleavage into small molecule amino acids is greatly increased. In the control example, there was only one freeze-thaw, and the degree of release was limited. The amino acids inside the cells were still trapped in the cells that were not completely ruptured and were difficult to release.

[0145] Different settings of melting temperature are also crucial. Low-temperature melting in the embodiment can gently activate the activity of proteases, slowly decompose proteins into free amino acids, and avoid enzyme inactivation caused by high-temperature melting. Although high-temperature melting in the comparative example speeds up the extraction speed, it causes some proteases to lose activity and the amount of amino acid release is limited. Higher melting temperatures may also cause amino acid degradation, which further affects the extraction effect.

[0146] Centrifugation and ultrafiltration also play an important role. The high-precision centrifugation in the embodiment removes macromolecular impurities, creating conditions for the enrichment of amino acids. The molecular weight cutoff of the ultrafiltration membrane is set within a reasonable range, and small molecule amino acids are fully enriched. In the comparative example, due to the unreasonable separation technology parameters, macromolecular impurities are mixed in, diluting the concentration of amino acids. In the end, the extraction efficiency shown by the embodiment far exceeds that of the comparative example.

[0147] Experiment 4: Extraction efficiency experiment Sample preparation: According to the processes of Examples 1, 2, 3 and Comparative Examples 1, 2, 3, extracts of Gecko foetida, Pheretima lumbricoides and Agkistrodon halys were prepared respectively.

[0148] Take 10 mL of each sample, record the initial liquid mass, and store at 4°C for later use.

[0149] Extract quality determination: Transfer 10 mL of the extract to a 105 °C oven and dry to constant weight.

[0150] Use an electronic balance to record the dry mass of the residue to two decimal places.

[0151] Calculate the extraction efficiency: extract mass (mg) / raw material mass (g) × 100%.

[0152] Experimental setup: Each sample was measured twice and the average value was taken.

[0153] Each group of experiments was performed independently to avoid cross contamination.

[0154] Table 4 Extraction efficiency test results Sample No. Initial mass of extract (g) Residue mass (mg) Extraction efficiency (%) Example 1-1 10.02 92.8 9.26 Example 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 Example 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 The data comparison of extraction efficiency makes it clear at a glance. In the examples, through optimized freeze-thaw and separation technology, the extraction efficiency is generally close to 9% or more, which is significantly better than the 6-7% of the comparative example. This is not a simple numerical difference, but a reflection of the process design itself. The examples were repeatedly frozen and thawed, and after the cell membrane was mechanically damaged by ice crystals, the potential for the release of effective ingredients was fully tapped. In the comparative example, there was only one freeze-thaw, the degree of cell wall disruption was limited, and the extraction efficiency was naturally suppressed at a low level. Such a result was within expectations.

[0155] In addition to the number of freeze-thaw cycles, melting conditions are also a key factor in determining efficiency. The embodiment selected a lower melting temperature, which can effectively activate enzyme activity and allow proteins and peptides to further degrade into small molecules that are easier to release. The high-temperature melting of the comparative example is unfavorable both in terms of cell penetration and the stability of active ingredients. Some ingredients may even aggregate or degrade due to high temperature, which directly reduces the quality of the extract. The experimental data confirms this point well.

[0156] The contribution of separation technology cannot be ignored. In the examples, the parameters of centrifugation and ultrafiltration were precisely adjusted so that high-molecular impurities were efficiently separated, leaving behind a purified high-concentration extract. However, the rough separation of the comparative example failed to effectively remove the macromolecular interfering components. Ultimately, this difference in separation efficiency directly affects the purity and total amount of the extract.

[0157] Experiment 5: Final product moisture content experiment Sample preparation: According to the processes of Example 4 and Comparative Example 4, dry powders of Agkistrodon acutus extract were prepared respectively.

[0158] 2 g of samples were randomly taken from each group of products for moisture content determination.

[0159] The samples were stored in sealed bags to avoid the influence of external humidity.

[0160] Determination method: Instrument and setup: Karl Fischer titrator (Mettler Toledo V20) was used.

[0161] The titration solvent was anhydrous methanol, and the concentration of the Karl Fischer reagent titration solution was 5 mg / mL.

[0162] Steps: The samples were added to a microwave digestion vessel and heated to 60 °C to release water.

[0163] During the titration the water content released from the sample is determined by potentiometric titration with Karl Fischer reagent.

[0164] Data Records: Each sample was measured twice and the average value was recorded.

[0165] Experimental setup: A comparison group (comparative example) and an experimental group (example) were set up to detect the control effect of different processes on the moisture content.

[0166] The ambient temperature was maintained at 25°C and the humidity was controlled below 40%.

[0167] Table 5 Test results of moisture content of dry powder in Examples and Comparative Examples Sample No. 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 The comparison of experimental data clearly shows that the moisture content of the dry powder in the embodiment is stably controlled below 6%, while the moisture content of the comparative example is close to 11%, which is obviously twice as high. This result is directly attributed to the accuracy of the freeze-drying process. The low-temperature pre-freezing and vacuum sublimation drying in the embodiment effectively avoids the residual moisture inside the sample through a gentle water sublimation process. In the comparative example, the sublimation temperature is high and the vacuum control is insufficient, resulting in the inability to completely remove the moisture, and the final drying effect is significantly inferior.

[0168] Moisture content is crucial to the long-term stability of the product. The low moisture content in the dry powder of the example reduces the possibility of microbial growth and oxidation reaction, while increasing the shelf life. In contrast, the high moisture content of the comparative example is not only susceptible to moisture and agglomeration, but may also produce odor or become ineffective due to microbial action during storage. This difference fully demonstrates the profound impact of precise control of freeze-drying technology on product quality.

[0169] The temperature setting and time arrangement of the drying process also directly determine the moisture content. The embodiment uses staged pre-freezing, sublimation and analytical drying to make the moisture release more thorough and avoid the destruction of components caused by high temperature. In the comparative example, due to the unreasonable adjustment of the drying parameters, the moisture release process was interrupted and the residual moisture remained in the sample after drying.

[0170] Experiment 6: Product stability test Sample preparation: According to the processes of Example 1 and Comparative Example 1, dry powders of Gecko extract were prepared respectively.

[0171] Take 5g of sample from each group, pack them into aluminum foil bags, and seal them as test samples.

[0172] Set storage conditions: room temperature (25℃±2℃) and high temperature (40℃±2℃).

[0173] Detection method: Protein content detection: The protein content of each sample was determined using the Coomassie Brilliant Blue method according to the method of Experiment 1, and the monthly data changes were recorded.

[0174] Free amino acid content detection: The free amino acid content was determined using an automatic amino acid analyzer according to the method of Experiment 3.

[0175] Total extract mass: 2 g of samples were taken every month, and the mass of the residue after drying was determined according to the method of Experiment 4, and the total amount of extract was calculated.

[0176] Experimental setup: Each group of samples was tested once a month for 6 consecutive months.

[0177] To reduce environmental interference, the storage environment humidity should be controlled below 40%.

[0178] Table 6 Product stability test results (room temperature storage for 6 months) Sample No. month Protein content (mg / g) Free amino acid content (mg / g) Extract mass (%) Example 1-1 0 month 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 0 month 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 The results are clear at a glance. The decrease in protein and free amino acid content of the product of the embodiment at room temperature is much smaller than that of the comparative example, and the quality of the extract remains stable. Thanks to the precise control of freeze-drying in the embodiment, the moisture content of the dry powder is low, and the oxidative degradation reaction is almost suppressed. The high-moisture product of the comparative example is more susceptible to moisture absorption during storage, protein oxidation is accelerated, free amino acid decomposition is significant, and the quality deterioration rate is significantly faster. These changes intuitively prove the decisive role of different drying processes on the stability of the final product.

[0179] The unique advantage of freeze-drying technology lies not only in efficient dehydration, but also in the protection of the structure of active ingredients during the gentle sublimation process. The process conditions of the embodiment are designed just right, which not only avoids the damage of high temperature to proteins and amino acids, but also minimizes the residual moisture in the product. However, in the comparative example, due to the high sublimation temperature, some proteins have denatured and the stability has been greatly reduced. This unreasonable process setting directly leads to quality loss during the storage stage.

[0180] The oxidation reaction is particularly active under high humidity conditions. The free amino acid content of the comparative example decreased significantly in the experiment, which is closely related to the high moisture content. The dried product of the embodiment shows extremely high stability due to low moisture content, and its antioxidant capacity is obviously far superior to that of the comparative example.

[0181] Experiment 7: Extraction experiment of medicinal effect related components Sample preparation: The extracts of Eupolyphaga sinensis were prepared according to the processes of Example 5 and Comparative Example 5.

[0182] Take 10 mL of the extract from each group, dilute it to 50 mL with cold distilled water, filter it and set aside.

[0183] Detection method: Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: Chromatographic column: C18 reverse phase column (150×4.6 mm, 5 μm particle size).

[0184] Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile.

[0185] Gradient elution: Initially 95% A, 5% B, linear gradient to 50% A, 50% B within 5 minutes, hold for 5 minutes.

[0186] Flow rate: 0.3 mL / min; column temperature: 30°C.

[0187] Target ingredients: Small molecule proteins: Detect targets with a molecular weight range of 1kDa-5kDa through characteristic ion pairs.

[0188] Free amino acids: Quantitative analysis of total free amino acid concentrations by comparing with the standard curve.

[0189] Injection volume: 20 μL was injected each time and analyzed continuously. Each group of samples was measured twice.

[0190] Experimental setup: Maintain room temperature (25°C) to avoid secondary degradation of the sample due to high temperature.

[0191] Data were collected automatically by the instrument, and peak areas and target component concentrations were analyzed using standardized software.

[0192] Table 7 Test results of efficacy-related components of Examples and Comparative Examples The experiment shows that the concentration of small molecule proteins and free amino acids extracted in the embodiment are significantly higher than those in the comparative example, and this difference is by no means accidental. The freeze-thaw extraction process in the embodiment, through repeated freeze-thaw cycles, the mechanical destruction of the cell membrane by ice crystals is extremely thorough, so that the small molecule proteins in the cells can be released smoothly. However, the comparative example only underwent one freeze-thaw cycle, and the release efficiency was greatly reduced. Many active ingredients are still retained in the unbroken cells, which directly limits the effectiveness of the extraction.

[0193] The reasonable setting of the melting temperature is also key. The low-temperature melting condition of the embodiment not only avoids the secondary damage of high temperature to small molecule proteins, but also retains the activity of the enzyme, so that the effective ingredients are fully released. However, at the higher melting temperature in the comparative example, the enzyme activity has already dropped significantly, and some small molecule proteins may even aggregate or denature due to overheating. Such differences in process details make the concentrations of small molecule proteins and free amino acids in the extract of the comparative example lower than those in the embodiment.

[0194] In addition, the enrichment effect of separation technology on the target component cannot be ignored. The embodiment uses precise centrifugation and ultrafiltration separation to effectively remove high molecular impurities and further concentrate small molecular proteins. In the comparative example, the separation process is not refined, and more high molecular impurities remain, diluting the concentration of active ingredients.

[0195] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for preparing fresh animal medicine, characterized in that: The following steps are involved: Step (1) cleaning: remove the non-medicinal parts of the fresh animal medicine, cut into pieces and clean; Step (2) low temperature frozen storage: the cleaned fresh animal drug is placed in a frozen state at -15°C to -25°C; 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; Step (4) freeze-thaw extraction: freeze the homogenized material at -25°C ± 2°C for 20-24 hours, and then thaw it at 37°C ± 1°C for 20-24 hours, repeating 2-4 times; Step (5) fractionation: subjecting the frozen-thawed material to low-speed centrifugation, high-speed centrifugation and ultrafiltration separation in sequence; Step (6) freeze drying: the separated liquid is freeze dried at low temperature, wherein the pre-freezing temperature is below -35°C, the sublimation temperature is between -20°C and -5°C, and the drying temperature is 35°C ± 2°C.

2. A process for preparing fresh animal medicine according to claim 1, characterized in that: The homogenization process in step (3) comprises: Add distilled water or sterile water in a 1:1 ratio during the homogenization process; The homogenization time is 20-40 minutes.

3. A process for preparing fresh animal medicine according to claim 1, characterized in that: The freeze-thaw extraction in step (4) comprises: During the freeze-thaw process, the material is frozen for 20-24 hours under freezing conditions; During the thawing stage, the material is thawed in a water bath for 20-24 hours, and the number of freeze-thaw cycles is 3 times.

4. A process for preparing fresh animal medicine according to claim 1, characterized in that: The low-speed centrifugation in step (5) includes the following conditions: The centrifuge speed is 3000-7000rpm, and the time is 10-20 minutes; The centrifugation was performed three times, and the flow rates of the drug solution were controlled at 15 L / min, 12 L / min, and 10 L / min, respectively.

5. A process for preparing fresh animal medicine according to claim 1, characterized in that: The high-speed centrifugation in step (5) includes the following conditions: The speed of the high-speed centrifuge is 8000-12000rpm, and the time is 5-10 minutes; The flow rate of the drug solution is below 5L / min.

6. A process for preparing fresh animal medicine according to claim 1, characterized in that: The ultrafiltration separation in step (5) comprises: Use ultrafiltration membrane with a molecular weight cutoff of less than 50,000; The filtration flow rate is 1-3 L / min, and the filtration temperature is controlled below 19°C.

7. A process for preparing fresh animal medicine according to claim 1, characterized in that: The freeze drying in step (6) includes the following conditions: The pre-freezing time is 1-3 hours, and the pre-freezing temperature is below -35°C; Sublimation drying time is 6-12 hours, and the vacuum degree is below 35Pa; The moisture content of the final product during the drying process is ≤6%.

8. A process for preparing fresh animal medicine according to claim 1, characterized in that: The volatile alkaline substance content of the fresh animal medicine in the cleaning step is no more than 33 mg / 100 g in terms of nitrogen.

9. A process for preparing fresh animal medicine according to claim 1, characterized in that: Each treatment from step (3) to step (6) is carried out in an environment with a temperature not higher than 19°C.

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