Extraction method of a water bear polypeptide and application thereof in freeze-drying protective agent

Abstract

The application provides an extraction method of water bear polypeptide and application thereof in freeze-drying protectant, and belongs to the technical field of freeze-drying protectant. First, frozen worm bodies are prepared, the frozen worm bodies are mixed with pre-cooled phosphate buffer, and then are subjected to crushing homogenization, leaching and second centrifugation in sequence, so that polypeptide crude extract is obtained through low-temperature mild extraction; then, under stirring, ammonium sulfate solution is added dropwise into the polypeptide crude extract, and low-temperature standing and low-temperature centrifugation are sequentially performed, so that refined extract is obtained; then, ultrafiltration treatment and dialysis are sequentially performed, so that the purified polypeptide solution is obtained through fractional impurity removal; finally, the purified polypeptide solution is subjected to low-temperature reduced-pressure concentration and vacuum freeze-drying / vacuum drying in sequence, so that high-activity water bear polypeptide is obtained through ultrafiltration desalting, and the high-activity water bear polypeptide is used as a core component to construct an efficient freeze-drying protectant, so that the efficient freeze-drying protectant is used for efficient freeze-drying stabilization of biological products such as live vaccines, probiotics, enzyme preparations and diagnostic reagents.

Description

Technical Field

[0001] This invention relates to the field of freeze-drying protectants, and in particular to a method for extracting tardigrade polypeptides and their application in freeze-drying protectants. Background Technology

[0002] Freeze-drying technology, with its low-temperature vacuum dehydration characteristics, can preserve the structure and function of bioactive substances (such as live vaccines, probiotics, and enzyme preparations) to the greatest extent. It is currently the core method for long-term preservation of bioactive substances and is widely used in many fields such as biomedicine, food processing, and biochemicals. However, in actual freeze-drying processes, samples undergo a series of complex processes, including ice crystal formation, water sublimation, deep dehydration, and low-temperature phase transitions, which can easily trigger multiple damage effects, severely impairing the activity of bioactive substances. Specifically, ice crystal growth can puncture cell membranes and protein structures, dehydration stress can lead to the breakage of hydrogen bonds and structural collapse of biomolecules, and temperature fluctuations during freeze-drying can cause thermal denaturation of active substances. The combined effect of these multiple factors can result in a significant decrease in bioactivity. For example, the viral titer of Newcastle disease virus can decrease by 1-3 log after freeze-drying, significantly reducing the potency and efficacy of biological products and limiting the quality and shelf life of freeze-dried biological products.

[0003] To mitigate bioactivity damage during freeze-drying, the industry commonly employs the addition of freeze-drying protectants to stabilize biological results and maintain bioactivity. However, current mainstream protectant systems all suffer from significant technical flaws, making it difficult to balance protective efficacy, safety, and process compatibility. Sugar protectants such as trehalose and sucrose are currently the most widely used basic protectants, but their overall protective efficiency is relatively low, requiring high concentrations of 5% to 10% to achieve a basic protective effect. This not only increases product costs but also leads to problems such as easy crystallization and poor formability at low temperatures, potentially causing cracking and loose structure in freeze-dried products, thus affecting product stability. Protein protectants such as gelatin and bovine serum albumin (BSA), while possessing some biocompatibility, have extremely poor heat resistance and are prone to degradation and inactivation during freeze-drying and subsequent storage, resulting in poor sustainability of the protective effect. Furthermore, the raw material costs are high, and there is a potential risk of immunogenicity, which can easily trigger adverse reactions in biological applications. Furthermore, while chemically synthesized cryoprotectants such as glycerol and dimethyl sulfoxide (DMSO) can help improve freeze-drying effects, they possess a certain degree of toxicity, can interfere with the activity of biomolecules, and leave residues that are difficult to completely remove, significantly limiting their application in high-end biopharmaceuticals and food-grade biological products. In summary, the current lack of an efficient, safe, low-cost, and highly adaptable freeze-drying protective medium is a key technological bottleneck restricting the high-quality development of the freeze-dried biological products industry. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting tardigrade polypeptides and their application in freeze-drying protectants. The tardigrade polypeptides extracted by the method of this invention have strong thermal stability, high purity, few impurities, and clear reconstitution. Furthermore, as a freeze-drying protectant, it has the advantages of high protection efficiency, low dosage, good safety, strong biocompatibility, simple process, low cost, and easy large-scale production.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for extracting polypeptides from tardigrades, comprising the following steps: The tardigrades were washed, centrifuged, and frozen sequentially to obtain frozen tardigrades. The frozen insect bodies and pre-cooled phosphate buffer were mixed and then subjected to homogenization, extraction and centrifugation in sequence to obtain crude polypeptide extract. Under stirring conditions, ammonium sulfate solution was added dropwise to the crude polypeptide extract, followed by low-temperature standing and low-temperature centrifugation to obtain a refined extract. The refined extract was subjected to ultrafiltration and dialysis sequentially to obtain a purified polypeptide solution. The purified polypeptide solution was subjected to low-temperature vacuum concentration and vacuum freeze-drying / vacuum drying in sequence to obtain tardigrade polypeptide.

[0006] Preferably, the freezing temperature is -25℃ to -18℃, and the freezing time is 18 to 45 minutes.

[0007] Preferably, the temperature of the pre-cooled phosphate buffer is 3~5℃, the concentration of the pre-cooled phosphate buffer is 0.05mol / L, the pH of the pre-cooled phosphate buffer is 6.5, and the pre-cooled phosphate buffer contains 0.05~0.2 mmol / L PMSF protease inhibitor.

[0008] Preferably, the temperature of the homogenized slurry is 3~5℃, the rotation speed of the homogenized slurry is 7500~13000 rpm, and the homogenization time is 2~6 min.

[0009] Preferably, the ammonium sulfate solution is a saturated ammonium sulfate solution; the ammonium sulfate solution is added dropwise until the saturation of ammonium sulfate in the system is 25-35%.

[0010] Preferably, the vacuum freeze-drying conditions include: first, pre-freeze-drying at -45~-35℃ for 1~3h, then sublimation at -35~-28℃ and a vacuum of 10 Pa for 4~8h, followed by desorption drying at 20~28℃ and a vacuum of 10 Pa for 3~5h.

[0011] The present invention also provides the application of tardigrade polypeptides extracted by the extraction method described above in freeze-drying protectants.

[0012] Preferably, the freeze-drying protectant is used for the freeze-drying stability of live vaccines, probiotics, enzyme preparations, and diagnostic reagents.

[0013] Preferably, when the lyophilization protectant is used for the lyophilization stabilization of live vaccines, the lyophilization protectant is prepared from the following raw materials: tardigrade polypeptide, trehalose, magnesium sulfate heptahydrate, and phosphate buffer.

[0014] Preferably, when the freeze-drying protectant is used for the freeze-drying stability of probiotics, the freeze-drying protectant is prepared from the following raw materials: tardigrade polypeptide, sucrose, skim milk powder, sodium ascorbate, and deionized water.

[0015] The tardigrade polypeptides prepared by the extraction method provided by this invention can form an amorphous glassy state upon freeze-drying, encapsulating the biomolecules and preventing them from entering the interior of viruses / bacteria. They exert their protective effect through mechanisms such as water substitution, space filling, and antioxidation, exhibiting significantly higher protective efficiency than traditional protective agents. Furthermore, the extraction method provided by this invention has the following advantages: (1) The activity of tardigrade polypeptides is fully preserved, and the heat resistance is strong. Tardigrade polypeptides can withstand temperatures up to 100℃ with almost no loss of activity, and the protective ability is significantly better than that of traditional sugar and protein protectants; (2) Tardigrade polypeptides have high purity and few impurities, and their reconstitution is clear. This invention effectively removes impurities such as starch, polysaccharides, oils, and salt ions through low-temperature water extraction, ammonium sulfate fractionation precipitation, and ultrafiltration desalting. The purity of tardigrade polypeptides can reach more than 70%, without turbidity and with fast reconstitution speed, solving the key defect of difficult filtration; (3) Tardigrade polypeptides have high protective efficiency, low dosage, and excellent freeze-drying effect as a freeze-drying protectant. High-efficiency protection can be achieved at low concentrations (0.02%~0.05%), and the titer loss of Newcastle disease live vaccine after freeze-drying is ≤0.3%. log, the survival rate of freeze-dried probiotics can reach more than 85%; the freeze-dried appearance is loose and full, without collapse or shrinkage, and the stability is significantly improved; (4) the safety of tardigrade polypeptides is good, the biocompatibility is strong and the application range is wide. Tardigrade polypeptides are natural disordered proteins, non-toxic, non-immunogenic and do not enter the virus / bacteria. They are protected only by external water replacement, vitrification and anti-ice crystal mechanism. They can be widely used in biological products such as live vaccines, probiotics, enzyme preparations and diagnostic reagents; (5) the extraction process is simple, low cost and easy to scale up. The whole process is a mild physical process, without complicated equipment, high temperature and chemical modification. The extraction yield is stable at more than 2.0%, which is suitable for industrial scale-up and has good industrialization prospects. Attached Figure Description

[0016] Figure 1 This is an SDS-PAGE electrophoresis image of the tardigrade polypeptide prepared in Example 1 of this invention, wherein... Figure 1 The M on the left is a protein molecular weight standard. Figure 1 The right side contains tardigrade polypeptide powder; Figure 2 The SDS-PAGE electrophoresis band grayscale peak image of the tardigrade polypeptide prepared in Example 1 of this invention; Figure 3 A photograph of the tardigrade polypeptide / Newcastle disease virus freeze-dried formulation prepared in Application Example 1 of this invention. Figure 4 This is a comparison of the Newcastle disease virus titer results of different stages of the present invention, including the Newcastle disease virus stock solution, the tardigrade polypeptide / Newcastle disease virus freeze-dried formulation, and the tardigrade polypeptide / Newcastle disease virus freeze-dried formulation after 7 days of accelerated processing at 37°C. Figure 5 The images show the colony counting plates of the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months in Example 2 of the present invention. From left to right, the images show the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months. Figure 6 This is a graph showing the changes in viable bacteria count and survival rate before freeze-drying, after freeze-drying, and during storage at room temperature (25°C) for 6 months in Application Example 2 of this invention. Figure 7 This is a photograph of the sample after reconstitution of the tardigrade polypeptide / Lactobacillus plantarum dry preparation prepared in Application Example 2 of the present invention. Figure 8 This is a comparison chart of the changes in enzyme activity and relative activity retention rate of the lyophilized tardigrade polypeptide / α-amylase preparation in Example 4 of the present invention; Figure 9 This is a comparison chart of the heat resistance activities of α-amylase before freeze-drying, freeze-dried tardigrade polypeptide / α-amylase preparation after freeze-drying, and freeze-dried tardigrade polypeptide / α-amylase preparation after heat resistance at 60°C for 30 min in Example 4 of the present invention. Figure 10 This is a comparison of the Newcastle disease virus (NDV) LaSota strain liquid titer results of different stages of the present invention, including the trehalose / NDV freeze-dried formulation (i.e., the traditional trehalose protectant 5% shown in the figure) and the trehalose / NDV freeze-dried formulation after 7 days of accelerated processing at 37°C. Figure 11 This is a comparison of the Newcastle disease virus titer results of different stages of the present invention: Newcastle disease virus stock solution, BSA / Newcastle disease virus freeze-dried preparation (i.e., BSA protein (3%) protectant in the figure), and BSA / Newcastle disease virus freeze-dried preparation sample accelerated at 37°C for 7 days. Figure 12 This is a diagram showing the color change of BSA aqueous solution upon contact with iodine in this invention; Figure 13This is a comparison of the titer results of Newcastle disease virus stock solution, peptide-free Newcastle disease virus freeze-dried formulation (i.e., peptide-free protectant in the figure), and peptide-free Newcastle disease virus freeze-dried formulation after 7 days of accelerated processing at 37°C. Figure 14 This is a comparison of the titer results of Newcastle disease virus stock solution, virus lyophilization agent (i.e., the blank control without protectant in the figure), and virus lyophilization agent sample after 7 days of accelerated drying at 37°C in different stages of this invention. Figure 15 The images show the colony counting plates of the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months in Comparative Example 5 of this invention. From left to right, the images show the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months. Figure 16 This is a graph showing the changes in viable bacteria count and survival rate in Comparative Example 5 of the present invention before freeze-drying, after freeze-drying, and during storage at room temperature of 25°C for 6 months. Figure 17 The images show the colony counting plates of the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months in Comparative Example 6 of this invention. From left to right, the images show the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months. Figure 18 This is a graph showing the changes in viable bacteria count and survival rate in Comparative Example 6 of the present invention before freeze-drying, after freeze-drying, and during storage at room temperature of 25°C for 6 months. Figure 19 The images show the colony counting plates of the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months in Comparative Example 7 of this invention. From left to right, the images show the samples before freeze-drying, after freeze-drying, and after being sealed and stored at 25°C for 6 months. Figure 20 This is a graph showing the changes in viable bacteria count and survival rate in Comparative Example 7 of the present invention before freeze-drying, after freeze-drying, and during storage at room temperature of 25°C for 6 months. Figure 21 This is a comparison chart of the changes in enzyme activity and relative activity retention rate of the BSA / α-amylase lyophilized formulation (i.e., the BSA protectant in the figure) in Comparative Example 8 of the present invention. Figure 22 This is a comparison of the heat resistance activities of α-amylase before freeze-drying, the freeze-dried BSA / α-amylase preparation after freeze-drying (i.e., the BSA group in the figure), and the freeze-dried BSA / α-amylase preparation after heat resistance at 60°C for 30 min in Comparative Example 8 of the present invention. Figure 23 This is a comparison chart of the changes in enzyme activity (i.e., BSA protectant) and relative activity retention rate of the α-amylase lyophilized preparation in Comparative Example 9 of the present invention. Figure 24This is a comparison chart of the heat resistance activities of α-amylase before freeze-drying, the freeze-dried α-amylase preparation after freeze-drying (i.e., the one without peptide protectant shown in the figure), and the freeze-dried α-amylase preparation after heat resistance at 60°C for 30 min. Detailed Implementation

[0017] This invention provides a method for extracting polypeptides from tardigrades, comprising the following steps: The tardigrades were washed, centrifuged, and frozen sequentially to obtain frozen tardigrades. The frozen insect bodies and pre-cooled phosphate buffer were mixed and then subjected to homogenization, extraction and centrifugation in sequence to obtain crude polypeptide extract. Under stirring conditions, ammonium sulfate solution was added dropwise to the crude polypeptide extract, followed by low-temperature standing and low-temperature centrifugation to obtain a refined extract. The refined extract was subjected to ultrafiltration and dialysis sequentially to obtain a purified polypeptide solution. The purified polypeptide solution was subjected to low-temperature vacuum concentration and vacuum freeze-drying / vacuum drying in sequence to obtain tardigrade polypeptide.

[0018] Unless otherwise specified, all raw materials used in this invention are commercially available products in the art.

[0019] The present invention involves washing, centrifuging, and freezing the tardigrades in sequence to obtain frozen tardigrades.

[0020] In this invention, the tardigrades are preferably dried dormant tardigrades (water content ≤8wt%) or fresh live tardigrades, more preferably dried dormant tardigrades. The tardigrades used in this invention are free from mold, impurities, and microbial contamination. The dried dormant tardigrades used in this invention have a higher content and more stable activity of endogenous disordered polypeptides (CAHS / TDPs), making them a preferred raw material. In this invention, the washing temperature is preferably 2-6℃. In this invention, the washing includes adding 4℃ pre-cooled deionized water to the tardigrades at a material-to-liquid ratio of 1g:5mL, and stirring and washing three times, each time for 5 minutes. This invention removes mud, surface attachments, and free microorganisms through washing, and controls the washing temperature to avoid the easy growth of microorganisms and degradation of polypeptides during room temperature washing. In this invention, the temperature of the first centrifugation is preferably 2-6℃, more preferably 4℃; the speed of the first centrifugation is preferably 3000-5000 rpm; and the time of the first centrifugation is preferably 3-10 minutes, more preferably 5 minutes. This invention involves discarding the supernatant after the first centrifugation and collecting the precipitate of the insect body, while avoiding excessively high centrifugation speeds that could break the insect body and introduce impurities. In this invention, the freezing temperature is preferably -25℃ to -18℃, more preferably -20℃; the freezing time is preferably 18 to 45 minutes, more preferably 30 minutes. By controlling the freezing temperature and time, this invention softens the cell walls of the insect body, facilitating subsequent gentle fragmentation and avoiding high-temperature denaturation, structural damage, and decreased activity of peptides caused by room-temperature mechanical fragmentation. It also avoids insufficient fragmentation due to excessively short freezing times and damage to the peptide structure by ice crystals due to excessively long freezing times.

[0021] After obtaining the frozen insect bodies, the present invention mixes the frozen insect bodies with pre-cooled phosphate buffer, and then sequentially performs homogenization, extraction, and centrifugation to obtain crude polypeptide extract.

[0022] In this invention, the temperature of the pre-cooled phosphate buffer is preferably 3-5°C, the concentration of the pre-cooled phosphate buffer is preferably 0.05 mol / L, the pH of the pre-cooled phosphate buffer is 6.5, and the pre-cooled phosphate buffer contains 0.05-0.2 mmol / L PMSF protease inhibitor. This invention inhibits endogenous protease degradation of peptides by adding an appropriate amount of PMSF protease inhibitor to the pre-cooled phosphate buffer; the absence of PMSF protease inhibitor leads to a decrease in peptide content of more than 30%. In this invention, the material-to-liquid ratio of the frozen insect body to the pre-cooled phosphate buffer is preferably 1 g:(6-10) mL, more preferably 1 g:8 mL. This invention avoids incomplete peptide dissolution due to excessively low material-to-liquid ratio and excessive energy consumption for subsequent concentration by controlling the material-to-liquid ratio of the frozen insect body to the pre-cooled phosphate buffer. In this invention, the homogenization is preferably performed using a low-speed tissue homogenizer. In this invention, the temperature of the homogenization process is preferably 3-5°C, more preferably 4°C; the rotation speed of the homogenization process is preferably 7500-13000 rpm, more preferably 8000-12000 rpm; and the homogenization time is preferably 2-6 min, more preferably 3-5 min. This invention, by controlling the temperature, rotation speed, and time of homogenization, ensures that only the cell walls of the insect body are disrupted, without damaging the disordered structure of the polypeptides. In this invention, the extraction is preferably carried out in a static, sealed container; the extraction temperature is preferably 2-6°C, more preferably 4°C. In this invention, the extraction time is preferably 1-3 h, more preferably 2 h. In this invention, during the extraction process, gentle stirring is preferably performed once every 30 min. This invention promotes the full dissolution of water-soluble disordered peptides by controlling the extraction conditions, temperature, time, and gentle stirring every 30 minutes. It avoids extraction temperatures above 10°C, which can lead to peptide degradation and reduced activity; extraction temperatures below 0°C, which can cause the solution to freeze and render the extraction ineffective; extraction times that are too short result in insufficient dissolution; and extraction times that are too long introduce impurities and increase the risk of microbial contamination. In this invention, the preferred temperature for the second centrifugation is 2-6°C, more preferably 4°C; the preferred centrifugation speed is 7000-9000 rpm; and the preferred centrifugation time is 13-18 minutes, more preferably 15 minutes. After the second centrifugation, the bottom insect residue is discarded, and the supernatant is collected to obtain a crude peptide extract.

[0023] After obtaining the crude polypeptide extract, the present invention adds ammonium sulfate solution dropwise to the crude polypeptide extract under stirring conditions, and then performs low-temperature standing and low-temperature centrifugation in sequence to obtain the refined extract.

[0024] In this invention, the ammonium sulfate solution is preferably a saturated ammonium sulfate solution; the ammonium sulfate solution is added dropwise until the saturation of ammonium sulfate in the system is preferably 25%~35%, more preferably 28%~32%. This invention controls the saturation of ammonium sulfate in the system to avoid incomplete precipitation of impurities when the saturation is below 25%, and easy co-precipitation and decreased recovery rate of target peptides when the saturation is above 35%. In this invention, the temperature of the system during the addition of ammonium sulfate solution is preferably 2~6℃, more preferably 4℃. In this invention, the dropping rate of the ammonium sulfate solution is preferably 10~15 mL / min. This invention controls the dropping rate of the ammonium sulfate solution under stirring to avoid localized excessive salt concentration caused by rapid dropping, leading to uneven local salting out and peptide denaturation. In this invention, the temperature for low-temperature settling is preferably 2~6℃, more preferably 4℃; the settling time is preferably 0.5~1.5h. This invention controls the temperature and time of low-temperature settling to ensure sufficient precipitation of large-molecule proteins (such as structural proteins and enzyme proteins). This avoids insufficient precipitation due to excessively short settling times, and excessively long settling times that increase energy consumption without any gain. In this invention, the preferred temperature for low-temperature centrifugation is 2-6°C, more preferably 4°C; the preferred centrifugation speed is 4000-6000 rpm; and the preferred centrifugation time is 8-12 minutes, more preferably 10 minutes. After low-temperature centrifugation, the bottom precipitate is discarded, and the supernatant is collected to obtain a refined extract rich in small-molecule disordered polypeptides.

[0025] After obtaining the refined extract, the present invention sequentially subjected the refined extract to ultrafiltration and dialysis to obtain a purified polypeptide solution.

[0026] In this invention, the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is preferably 2-5 kDa, more preferably 3 kDa. The temperature of the ultrafiltration process is preferably 2-6°C, more preferably 4°C; the pressure is preferably 0.2-0.4 MPa. This invention removes ammonium sulfate ions, small molecule impurities (such as free amino acids, polysaccharide fragments), and pigments through ultrafiltration, and collects the filtrate to achieve ultrafiltration purification. This invention controls the molecular weight cutoff of the ultrafiltration membrane to avoid loss of target peptides below 2 kDa and inability to remove small molecule impurities above 5 kDa. It also controls the temperature of the ultrafiltration process to avoid membrane fouling and decreased peptide activity at room temperature. Finally, it controls the pressure to avoid low ultrafiltration efficiency due to excessively low pressure and membrane damage and impurity leakage due to excessively high pressure. In this invention, the dialysis is preferably performed using a 3 kDa dialysis bag at 2-6°C for 22-26 hours until the conductivity of the dialysate is <10 μS / cm, and the deionized water is replaced every 6 hours. This invention further removes trace salt ions through dialysis and dialyzes until the conductivity of the dialysate is <10 μS / cm, ensuring the purity of the peptides.

[0027] After obtaining the purified polypeptide solution, the present invention sequentially performs low-temperature vacuum concentration and vacuum freeze-drying / vacuum drying on the purified polypeptide solution to obtain tardigrade polypeptide. In this invention, the low-temperature vacuum concentration is preferably performed using a rotary evaporator; the low-temperature vacuum concentration temperature is preferably 40~60℃, more preferably 50℃; the low-temperature vacuum concentration is reduced to 1 / 4~1 / 6 of the original volume, more preferably 1 / 5. This invention controls the low-temperature vacuum concentration temperature to avoid the shrinkage and loss of activity of disordered peptide structures that easily occur above 60℃. In this invention, the vacuum drying temperature is preferably 35~45℃, more preferably 40℃. In this invention, the vacuum freeze-drying conditions include: first, pre-freeze-drying at -45~-35℃ for 1~3 hours, then sublimation at -35~-28℃ and a vacuum of 10 Pa for 4~8 hours, followed by desorption drying at 20~28℃ and a vacuum of 10 Pa for 3~5 hours; more preferably, it includes: first, pre-freeze-drying at -40℃ for 2 hours, then sublimation at -30℃ and a vacuum of 10 Pa for 6 hours, followed by desorption drying at 25℃ and a vacuum of 10 Pa for 4 hours. This invention, by controlling the conditions of vacuum freeze-drying, can maximize the preservation of the disordered structure and activity of peptides, ultimately obtaining a light yellow, loose powder of tardigrade peptides. This invention controls the vacuum drying temperature to ≤40℃ to avoid high-temperature damage. In this invention, the moisture content of the tardigrade peptides is ≤5wt%. By controlling the moisture content of the tardigrade peptides, this invention avoids excessive moisture absorption and deterioration, while insufficient moisture increases energy consumption and causes the peptides to clump. In this invention, the tardigrade peptides are preferably stored in a sealed, light-protected container at -20℃.

[0028] The method provided by this invention prepares tardigrade polypeptides (also known as tardigrade-derived disordered polypeptides, CAHS / TDPs) that are a light yellow, loose powder with no odor, no lumps, and no impurities. They consist of 80-120 amino acids, rich in hydrophilic amino acids such as glycine, serine, and threonine, with a disordered structure accounting for ≥90%. The purity is ≥70% (optimally 75%-85%). The molecular weight is 3-12 kDa (optimally 5-8 kDa). They have good solubility, readily soluble in water and phosphate buffer. After reconstitution, the solution is clear and transparent, without turbidity or precipitation. They exhibit good thermal stability; after boiling in a 100℃ water bath for 30 min, the activity retention rate is ≥95%. They are stable within a pH range of 5.0-8.0, with no significant decrease in activity. The water content of the tardigrade polypeptides is ≤5 wt%, and the yield is ≥2.0% (based on dry tardigrade weight).

[0029] This invention first prepares frozen tardigrades. The frozen tardigrades are mixed with pre-cooled phosphate buffer and then subjected to homogenization, extraction, and centrifugation to achieve low-temperature, gentle extraction of a crude polypeptide extract. Then, under stirring conditions, ammonium sulfate solution is added dropwise to the crude polypeptide extract, followed by low-temperature settling and centrifugation to obtain a refined extract. This refined extract is then subjected to ultrafiltration and dialysis to remove impurities and obtain a purified polypeptide solution. The purified polypeptide solution is then subjected to low-temperature vacuum concentration and vacuum freeze-drying / vacuum drying to achieve ultrafiltration desalting and obtain highly active tardigrade polypeptides. These polypeptides are used as core components to construct a highly efficient freeze-drying protectant for the efficient freeze-drying stabilization of biological products such as live vaccines, probiotics, enzyme preparations, and diagnostic reagents.

[0030] The present invention also provides the application of tardigrade polypeptides extracted by the extraction method described above in freeze-drying protectants.

[0031] In this invention, the freeze-drying protectant is preferably used for the freeze-drying stability of live vaccines, probiotics, enzyme preparations, and diagnostic reagents.

[0032] In this invention, the live vaccine preferably includes at least one of Newcastle disease live vaccine, infectious bursal disease live vaccine, classical swine fever live vaccine, and avian influenza live vaccine. In this invention, the probiotics preferably include at least one of Lactobacillus plantarum, lactic acid bacteria, Bifidobacterium, Escherichia coli, and Bacillus subtilis. In this invention, the enzyme preparation preferably includes at least one of α-amylase, protease, and lipase. In this invention, the diagnostic reagent preferably includes at least one of antibody detection kit and colloidal gold in vitro diagnostic test strip.

[0033] In this invention, when the lyophilization protectant is used for the lyophilization stabilization of live vaccines, the lyophilization protectant (also known as the lyophilization protectant for live vaccines) is prepared from the following raw materials: tardigrade polypeptide, trehalose, magnesium sulfate heptahydrate, and phosphate buffer.

[0034] In this invention, the concentration of tardigrade polypeptide in the lyophilized protectant for live vaccines is preferably 0.002~0.005 g / mL, more preferably 0.003 g / mL. In this invention, the concentration of trehalose is preferably 0.02~0.03 g / mL, more preferably 0.025 g / mL. In this invention, the concentration of magnesium sulfate heptahydrate is preferably 0.0005~0.0010 g / mL, more preferably 0.0008 g / mL. In this invention, the concentration of the phosphate buffer is preferably 0.05 mol / L; the pH of the phosphate buffer is preferably 6.8~7.2, more preferably 7.0. This invention controls the concentration of each component within the above-mentioned range, and employs a synergistic protection mechanism. Tardigrade polypeptides penetrate the bacterial cell membrane, protecting intracellular proteins and nucleic acids and resisting damage from freeze-drying low temperatures and dehydration. Trehalose forms a glassy film that encapsulates the bacterial cells, reducing the risk of ice crystals piercing the cells, and works synergistically with the polypeptides to lock in water and prevent denaturation. Magnesium sulfate heptahydrate regulates ionic strength, preventing protein aggregation and enhancing the binding ability of polypeptides to the cell membrane. Phosphate buffer stabilizes the pH of the system, ensuring that the bioactivity of polypeptides and antigens is not destroyed by acids or alkalis.

[0035] In this invention, the method for preparing the freeze-dried protectant for live vaccines preferably includes the following steps: slowly adding tardigrade polypeptide to phosphate buffer at 4°C, then stirring at low speed at 4°C until completely dissolved and the solution is clear; sequentially adding trehalose and magnesium sulfate heptahydrate, continuing to stir at low speed at 4°C for 10 min until completely dissolved; then adjusting the pH of the system to 6.8~7.2 with phosphate; filtering with a sterile filter membrane for sterilization; and storing in a sealed container at 4°C in the dark to obtain the freeze-dried protectant for live vaccines.

[0036] In this invention, the low-speed stirring speed is 200-300 rpm; the low-speed stirring time is 15-20 min. In this invention, the sterile filter membrane used for sterilization employs a sterile filter membrane with a pore size of 0.22 μm. In this invention, the shelf life of the freeze-drying protectant used for live vaccines is preferably 3 months.

[0037] In this invention, the method for using the freeze-dried protective agent for live vaccines to protect Newcastle disease live vaccines includes the following steps: Newcastle disease live vaccine virus solution (titer 10) 6.6 ~10 7.0 Mix TCID50 / mL with the lyophilized protectant for live vaccines at a volume ratio of 1:1, mix gently to avoid vigorous shaking that could inactivate the virus, and then dispense into 0.5 mL vials. The dispensed product was pre-frozen at 40℃ for 3 h, then sublimated at -35℃ and 10 Pa for 6 h, then desorbed at 30℃ and 10 Pa for 5 h, and finally kept at 30℃ for 2 h, vacuum-sealed, and stored at 2~8℃ in the dark to obtain the lyophilized preparation of tardigrade polypeptide / Newcastle disease virus.

[0038] The present invention uses a freeze-drying protectant for live vaccines to freeze-dry Newcastle disease live vaccine. After freeze-drying, the titer loss is ≤0.3 log, the appearance is loose and full, without collapse or shrinkage, the reconstitution time is ≤1 min, the shelf life at 2~8℃ is ≥18 months, and the titer does not decrease significantly after 7 days of accelerated freezing at 37℃.

[0039] In this invention, when the freeze-drying protectant is used for the freeze-drying stability of probiotics, the freeze-drying protectant (also known as a freeze-drying protectant for probiotics) is prepared from the following raw materials: tardigrade polypeptide, sucrose, skim milk powder, sodium ascorbate, and deionized water.

[0040] In this invention, the concentration of the tardigrade polypeptide is preferably 0.003~0.008 g / mL, more preferably 0.005 g / mL. In this invention, the concentration of the sucrose is preferably 0.02~0.04 g / mL, more preferably 0.03 g / mL. In this invention, the concentration of the skim milk powder is preferably 0.015~0.03 g / mL, more preferably 0.02 g / mL. In this invention, the concentration of the sodium ascorbate is preferably 0.0015~0.002 g / mL, more preferably 0.001 g / mL. This invention controls the concentration of each component within the above-mentioned range, and works synergistically to protect the probiotics. The tardigrade polypeptide repairs the cell membrane of the probiotics, reducing cell damage during freeze-drying and storage. The vitrification of sucrose protects the cells and alleviates the osmotic pressure shock during reconstitution, while the polypeptide enhances stability. The protein coating of the skim milk powder isolates oxygen, slowly releases the polypeptide, and prolongs the protection time of the cells. Sodium ascorbate scavenge free radicals in the system, preventing the polypeptide from oxidizing and inactivating the live bacteria.

[0041] In this invention, the method for preparing the lyophilization protectant for probiotics preferably includes the following steps: slowly adding tardigrade polypeptide to a phosphate buffer at 4°C, then stirring at low speed at 4°C until completely dissolved and the solution is clear; sequentially adding sucrose, skim milk powder, and sodium ascorbate; continuing to stir at low speed at 4°C for 10 min until completely dissolved; then adjusting the pH of the system to 6.8-7.2 with phosphate; filtering with a sterile filter membrane for sterilization; and storing in a sealed container at 4°C in the dark to obtain the lyophilization protectant for live vaccines. In this invention, the low-speed stirring speed is 200-300 rpm; the low-speed stirring time is 15-20 min. In this invention, the sterile filter membrane used for sterilization employs a sterile filter membrane with a pore size of 0.22 μm. In this invention, the shelf life of the freeze-drying protectant used for probiotics is preferably 3 months.

[0042] Furthermore, after freeze-drying probiotics with tardigrade polypeptides, the survival rate of freeze-dried probiotics is ≥85%, and the survival rate after 6 months of storage at room temperature is ≥80%. After freeze-drying enzyme preparations with tardigrade polypeptides, the enzyme activity retention rate after freeze-drying is ≥90%, and the stability at room temperature is significantly improved.

[0043] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0044] Unless otherwise specified, all experiments were repeated three times, and the results are expressed as averages.

[0045] Example 1 A method for extracting polypeptides from tardigrades, comprising the following steps: According to the material-liquid ratio of 1g:5mL, take 10g of dried dormant tardigrades (water content 7.5wt%), add 4℃ pre-cooled deionized water to wash 3 times, 5 min each time, then centrifuge at 4℃ and 4000 rpm for 5 min, discard the supernatant, collect the tardigrade precipitate, and then freeze at -20℃ for 30 min to obtain frozen tardigrades. Add 80 mL of 4℃, 0.05 mol / L phosphate buffer (pH 6.5, containing 0.1 mmol / L PMSF) to 10 g of the frozen insect body at a material-to-liquid ratio of 1 g: 8 mL. Then, use a low-speed tissue homogenizer at 4℃ and 10000 rpm for 4 min to homogenize and obtain a homogenate. The homogenate was transferred to a sealed container and allowed to stand at 4°C for 2 hours for extraction. The mixture was gently stirred every 30 minutes. Then, it was centrifuged for 15 minutes at 4°C and 8000 rpm. The insect residue at the bottom was discarded, and the supernatant was collected to obtain the crude polypeptide extract. Under stirring conditions, saturated ammonium sulfate was slowly added dropwise to the crude polypeptide extract at a rate of 12 mL / min until the ammonium sulfate saturation in the system reached 30%. The temperature was controlled at 4℃ during the dropwise addition process. After the dropwise addition was completed, the mixture was allowed to stand at 4℃ for 1 h. Then, it was centrifuged at 5000 rpm at 4℃ for 10 min. The precipitate was discarded, and the supernatant was collected to obtain a refined extract rich in small molecule disordered polypeptides. The refined extract was subjected to ultrafiltration through a 3 kDa ultrafiltration membrane (4℃, 0.3 MPa), the filtrate was collected, and deionized water was used for dialysis at 4℃ for 24 h using a 3 kDa dialysis bag. The deionized water was replaced every 6 h until the conductivity of the dialysate was <10 μS / cm, thus obtaining the purified polypeptide solution. The purified polypeptide solution was concentrated to 16 mL under reduced pressure at 50 °C using a rotary evaporator, and then freeze-dried under vacuum to obtain 0.23 g of pale yellow tardigrade polypeptide powder, with a yield of 2.3%. The conditions for vacuum freeze-drying are as follows: first, pre-freeze-drying is performed at -40℃ for 2 hours, then sublimation is performed at -30℃ and a vacuum of 10 Pa for 6 hours, followed by desorption drying at 25℃ and a vacuum of 10 Pa for 4 hours.

[0046] The purity of the tardigrade polypeptide prepared in Example 1 was determined by SDS-PAGE grayscale analysis. The SDS-PAGE electrophoresis image of the tardigrade polypeptide prepared in Example 1 is shown below. Figure 1 As shown, where Figure 1 The M on the left is a protein molecular weight standard. Figure 1 The right side contains tardigrade polypeptide powder. (From...) Figure 1 It can be seen that the tardigrade polypeptide powder sample on the right only has a single main band below the 10kDa Marker band, corresponding to a target tardigrade polypeptide molecular weight range of 5~8 kDa. There are few extraneous bands, and it is preliminarily determined that the sample is mainly concentrated in the target molecular weight range.

[0047] The tardigrade polypeptide prepared in Example 1 was quantitatively analyzed by gel electrophoresis gray-scale scanning. The gray-scale peak diagram of the SDS-PAGE electrophoresis bands of the tardigrade polypeptide prepared in Example 1 is shown below. Figure 2 As shown in Table 1 below, the statistical results of the peak area integration of each characteristic peak in the electrophoresis of tardigrade polypeptide powder are presented.

[0048] Table 1 shows the peak area integral statistics of each characteristic peak in the electrophoresis of the tardigrade polypeptide powder prepared in Example 1.

[0049] The peak areas of each characteristic peak in Table 1 above were normalized and integrally calculated. The characteristic peak No. 6 corresponds to the target tardigrade polypeptide band, and its peak area accounts for 77.4% of the total area. That is, the protein purity of the tardigrade polypeptide powder prepared in Example 1 was determined to be 77.4% by SDS-PAGE grayscale analysis.

[0050] Thermal stability: The tardigrade polypeptide prepared in Example 1 was prepared into a polypeptide aqueous solution of 3 mg / mL, boiled at 100°C for 30 min, and its activity retention rate was 96% as determined by the lactate dehydrogenase (LDH) protective activity assay. Solubility: Using deionized water, the tardigrade polypeptide prepared in Example 1 was prepared into a 1 wt% aqueous solution that was clear and transparent without turbidity or precipitate. Physicochemical characterization: To detect whether starch impurities are present, the tardigrade polypeptide prepared in Example 1 was prepared into a 1 wt% tardigrade polypeptide aqueous solution and iodine solution was added. The solution showed no pink / blue color development, confirming that it did not contain starch filler, indicating that the tardigrade polypeptide raw material prepared in Example 1 did not contain polysaccharide impurities that could damage the virus.

[0051] Example 2 A freeze-dried protective agent for live vaccines is prepared from the following raw materials: tardigrade polypeptide (as in Example 1), trehalose, magnesium sulfate heptahydrate, phosphate buffer solution with a concentration of 0.05 mol / L and a pH of 7, and phosphate.

[0052] The concentrations of each component in the freeze-dried protective agent used for the live vaccine are: 0.003 g / mL tardigrade polypeptide, 0.025 g / mL trehalose, and 0.0008 g / mL magnesium sulfate heptahydrate, with a concentration of 0.05 mol / L.

[0053] The preparation method of the above-mentioned lyophilized protectant for live vaccines is as follows: 0.3 g of tardigrade polypeptide is slowly added to a portion of 0.05 mol / L phosphate buffer (pH 7.0) at 4°C, and then stirred at low speed at 4°C until completely dissolved and the solution is clear. 2.5 g of trehalose and 0.08 g of magnesium sulfate heptahydrate are added sequentially, and stirring at low speed at 4°C is continued for 10 min until completely dissolved. The volume is adjusted to 100 mL with the remaining phosphate buffer, and the pH of the system is adjusted to 7 with phosphate. The solution is then filtered through a sterile filter membrane with a pore size of 0.22 μm for sterilization, and stored in a sealed container at 4°C in the dark to obtain the lyophilized protectant for live vaccines.

[0054] Application Example 1 The Newcastle disease live vaccine was freeze-dried using the freeze-drying protectant for live vaccines described in Example 2. The steps were as follows: Virus solution: titer 10 6.6 TCID 50 / mL of Newcastle disease LaSota strain was used as the virus fluid; Mixing and dispensing: Gently mix the virus solution and the lyophilized protective agent for live vaccines at a ratio of 1:1 (v / v), and then dispense into 0.5 mL vials. Freeze-drying: Pre-freeze at 40℃ for 3 h, then sublimate at -35℃ and vacuum degree of 10 Pa for 6 h, then desorb at 30℃ and vacuum degree of 10 Pa for 5 h, and finally keep warm at 30℃ for 2 h, vacuum stopper seal, and store at 2~8℃ in the dark to obtain tardigrade polypeptide / Newcastle disease virus freeze-dried preparation. The indicators of the tardigrade polypeptide / Newcastle disease virus freeze-dried formulation prepared in accordance with Example 1 were tested, including: freeze-dried appearance, reconstitution time, moisture content, titer after freeze-drying, and titer after 7 days of accelerated testing at 37℃.

[0055] Test results (1) Appearance of freeze-dried product: loose and full, without collapse or cracks. The actual picture of the freeze-dried preparation of tardigrade polypeptide / Newcastle disease virus prepared in Example 1 is shown below. Figure 3 As shown; (2) Reconstitution time: The tardigrade polypeptide / Newcastle disease virus freeze-dried preparation prepared in Example 1 was mixed with deionized water and reconstituted in 42 s. The reconstituted solution was clear and free of turbidity. (3) Moisture content: The moisture content of the tardigrade polypeptide / Newcastle disease virus freeze-dried preparation prepared in Example 1 was 1.8 wt%; (4) Use TCID 50 The detection methods were as follows: The titer of the Newcastle disease LaSota strain virus solution was determined separately. Newcastle disease LaSota strain original viral titer: 10 6.6 TCID 50 / mL; After lyophilization using Example 1, the titer was 10. 6.3 TCID 50 / mL, titer loss 0.3 log; 37℃ accelerates titer over 7 days: 10 6.1 TCID 50 / mL, with a relative decrease in titer of only 0.2 log after lyophilization; The comparison of Newcastle disease virus titers at different stages of Newcastle disease virus stock solution, tardigrade peptide / Newcastle disease virus lyophilized formulation, and tardigrade peptide / Newcastle disease virus lyophilized formulation after 7 days of accelerated treatment at 37℃ is shown in the figure below. Figure 4 As shown.

[0056] Based on the above test results, the tardigrade polypeptide / Newcastle disease virus freeze-dried formulation prepared using Application Example 1 of this invention exhibits good appearance, a loose freeze-dried structure, excellent reconstitution properties, rapid reconstitution speed, and a homogeneous and stable system after reconstitution. The finished product's moisture content is controlled at a low level, reducing the risk of virus inactivation during storage. The virus titer loss during the freeze-drying process was only 0.3 log, and after 7 days of accelerated high-temperature (37℃) drying, the virus titer decreased only slightly by 0.2 log. This demonstrates that the freeze-drying protection system used in this invention can effectively encapsulate and protect the Newcastle disease virus LaSota strain during freeze-drying preparation and short-term high-temperature storage, significantly reducing virus activity attenuation during processing and storage. This effectively improves upon the shortcomings of existing conventional freeze-drying processes, such as large virus titer loss and poor thermal stability of the finished product, thus enhancing the process applicability and storage stability of the Newcastle disease virus freeze-dried formulation.

[0057] Example 3 A freeze-drying protectant for probiotics, prepared from the following raw materials: tardigrade polypeptide, sucrose, skim milk powder, sodium ascorbate, and deionized water (as in Example 1); The concentrations of each component in the freeze-drying protectant for probiotics are: 0.005 g / mL tardigrade polypeptide, 0.03 g / mL sucrose, 0.02 g / mL skim milk powder, and 0.001 g / mL sodium ascorbate.

[0058] The preparation method of the above-mentioned lyophilization protectant for probiotics includes the following steps: 0.5 g of tardigrade polypeptide was slowly added to a portion of deionized water at 4°C, and then stirred at low speed at 4°C until completely dissolved and the solution was clear. 3 g of sucrose, 2 g of skim milk powder, and 0.1 g of sodium ascorbate were added sequentially, and stirring at low speed at 4°C was continued for 10 min until completely dissolved. The volume was then adjusted to 100 mL using the remaining deionized water at 4°C. The pH of the system was then adjusted to 7 with phosphoric acid. The solution was filtered through a sterile membrane for sterilization, and stored in a sealed container at 4°C away from light to obtain a lyophilized protectant for probiotics.

[0059] Application Example 2 The freeze-drying protection of *Lactobacillus plantarum* was carried out using the freeze-drying protectant for probiotics described in Example 3, and the steps were as follows: Bacterial culture: Lactobacillus plantarum, cultured under MRS to OD. 600 =0.7, centrifuged at 4℃ and 6000 rpm for 8 min, and resuspended in PBS to 10 8 CFU / mL; Dispensing: Mix the bacterial solution and the freeze-drying protectant for probiotics at a ratio of 1:1, and then dispense them. Freeze-drying: Freeze-drying was carried out according to the freeze-drying method of Application Example 1 to obtain the freeze-dried preparation of tardigrade polypeptide / Lactobacillus plantarum; The indicators of the tardigrade polypeptide / Lactobacillus plantarum dry preparation prepared in accordance with Example 2 were tested, including: the number of viable bacteria before and after freeze-drying, the number of viable bacteria after storage at room temperature (25℃) for 6 months, and the reconstitution performance.

[0060] Experimental results: (1) Detection of viable bacteria count during freeze-drying and room temperature storage Plate counts were performed on samples at three key time points: before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C. The resulting images of the plate counts for the samples before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C in Application Example 2 are shown below. Figure 5 The statistical results of viable bacterial concentration and cell survival rate at each stage are as follows: ① Before freeze-drying: viable count was 3.1 × 10⁻⁶ 8 CFU / mL, with the bacterial survival rate set at 100% (calculation baseline); ② After lyophilization: viable count was 2.88 × 10⁻⁶. 8 CFU / mL, lyophilized bacterial cell survival rate 92.9%; ③ Stored at 25℃ for 6 months: viable bacterial count 2.42×10⁻⁶. 8 The CFU / mL concentration represents a relative initial cell survival rate of 78.1% compared to the initial cell survival rate before lyophilization.

[0061] The changes in viable bacterial count and survival rate before and after freeze-drying, and after storage at 25°C for 6 months, are shown in the graph in Example 2. Figure 6 As shown.

[0062] (2) Reconstitution performance: The reconstitution test was conducted on the tardigrade polypeptide / Lactobacillus plantarum dry preparation prepared in Example 2. The results showed that the preparation could be rapidly dispersed and dissolved in water in 50 seconds, eventually forming a homogeneous bacterial solution. The bacterial solution system was uniform, with no visible clumps or agglomerations. The sample state after reconstitution is shown in the following figure. Figure 7 As shown.

[0063] (3) Results Analysis 1) Cell preservation effect of freeze-drying process: Using the original Lactobacillus plantarum solution before freeze-drying as the initial control, after treatment with the freeze-drying process of this invention, the viable cell count of the sample increased from 3.1 × 10⁻⁶. 8 CFU / mL decreased to 2.88 × 10⁻⁶ 8 With a CFU / mL concentration, the bacterial cell survival rate reached 92.9%, with only a small amount of viable bacteria lost. This indicates that the freeze-drying system in Example 2 can effectively alleviate the damage to bacterial cells caused by low temperature, vacuum, and ice crystal formation during the freeze-drying process, and has a good cell protection effect on Lactobacillus plantarum.

[0064] 2) Long-term storage stability at room temperature: After being stored in a sealed container at 25℃ for 6 months, the viable bacterial count of the sample remained at 2.42 × 10⁻⁶.8 With a CFU / mL concentration and a relative initial bacterial survival rate of 78.1%, the viable cell count decreased gradually during storage, demonstrating that the freeze-dried formulation has excellent room temperature storage stability. This reduces the limitations of cold chain transportation and storage, and expands the application scenarios of the product.

[0065] 3) Physical properties of the formulation: The lyophilized product has good reconstitution properties and can be quickly dispersed after adding water. The reconstituted bacterial solution is uniform and stable without clumping, which shows that the formulation has excellent compatibility and facilitates dilution, administration and other operations in subsequent application processes.

[0066] In summary, the freeze-dried Lactobacillus plantarum preparation prepared by this invention has the advantages of high cell freeze-drying survival rate, good room temperature storage stability and excellent reconstitution processing performance, and its application advantages are significant.

[0067] Example 4 The freeze-drying stability of the tardigrade polypeptide-protected industrial enzyme (α-amylase) prepared in Example 1 was tested. I. Experimental Methods Protection system: 10 mL of α-amylase (1000 U / mL) + 10 mL of tardigrade polypeptide solution (1 wt%), resulting in a final protection system with a tardigrade polypeptide concentration of 0.5 wt% and an α-amylase concentration of 500 U / mL. Freeze-drying: The protective system was dispensed and freeze-dried according to the freeze-drying method of Application Example 1 to obtain a freeze-dried preparation of tardigrade polypeptide / α-amylase.

[0068] The indicators for detecting the above-mentioned lyophilized tardigrade polypeptide / α-amylase preparations include: enzyme activity after lyophilization and residual enzyme activity after heat resistance at 60℃ for 30 min.

[0069] Test results (1) The protective effect of freeze-drying process on α-amylase activity The enzyme activity test results after lyophilization were as follows: the initial enzyme activity (control before lyophilization) and the enzyme activity (U / ml) of Example 4 group (after lyophilization) were 500 and 468, respectively. The retention rate of Example 4 group relative to the initial enzyme activity (baseline 100%) was 93.6%.

[0070] A biaxial composite plot was plotted using the detection data to obtain a comparison of the changes in enzyme activity and relative activity retention rate of the tardigrade polypeptide / α-amylase lyophilized formulation in Example 4, as shown in the figure below. Figure 8 As shown, this visually reflects the differences in enzyme activity values ​​and activity retention among the groups.

[0071] (2) Heat resistance test at 60℃ 1. Results of enzyme activity detection in the heat resistance test The freeze-dried tardigrade polypeptide / α-amylase samples from Example 4 were subjected to heat treatment at 60°C for 30 min. Using the enzyme activity after freeze-drying as a baseline, the residual enzyme activity after heat treatment was measured and the activity retention rate was calculated. The results were as follows: Example 4 group: enzyme activity after heat treatment was 395 U / mL, with a relative activity retention rate of 84.4% compared to the freeze-dried samples in this group. 2. Comparison of enzyme activity between heat-resistant and non-heat-resistant groups A side-by-side bar chart of enzyme activity before freeze-drying, after freeze-drying, and after heat resistance at 60℃ for 30 min was plotted, resulting in a comparative graph of the heat resistance activities of α-amylase before freeze-drying, freeze-dried tardigrade polypeptide / α-amylase preparation after freeze-drying, and freeze-dried tardigrade polypeptide / α-amylase preparation after heat resistance at 60℃ for 30 min in Example 4, as shown in the figure. Figure 9 As shown, the heat resistance of the samples can be compared visually.

[0072] (3) Results Analysis 1) Protective effect of freeze-drying process During freeze-drying, vacuum, low temperature, and ice crystal formation can easily disrupt the spatial conformation of α-amylase protein, causing irreversible loss of enzyme activity. Using 500 U / mL of the original solution before freeze-drying as the activity baseline, the enzyme activity retention rate after freeze-drying in Example 4 of this invention reached 93.6%, with only a small amount of activity loss; while the enzyme activity retention rate after freeze-drying in the conventional control group was only 62.4%, with significantly higher enzyme activity loss than the system of this invention. This demonstrates that the freeze-drying protection system of this invention can effectively buffer the structural damage to amylase during the freeze-drying process and retain the enzyme's catalytic activity to the maximum extent.

[0073] 2) High-temperature thermal stability After heat treatment at 60℃ for 30 min, the enzyme activity of both groups of samples decreased to varying degrees, but the stability between the groups was significantly different: the activity of the sample in Example 4 of this invention was 84.4% of that after freeze-drying, and the enzyme activity decayed slowly at high temperature; the activity of the control group was only 50.6% after heat treatment, and nearly half of the enzyme activity was inactivated under high temperature conditions.

[0074] The results show that the freeze-drying protection system in Example 4 of this invention not only has a good enzyme protection effect during the freeze-drying stage, but also improves the high temperature tolerance of the amylase product, broadens the application temperature range of the α-amylase preparation, and has better product storage and application stability.

[0075] (a) Comparative example of freeze-drying Newcastle disease virus liquid Comparative Example 1: Traditional Trehalose Protectant (5%) I. Experimental Methods Traditional trehalose protectant formulation: 5% trehalose + 0.05 mol / L phosphate buffer (pH 7.0); The traditional trehalose protectant is then mixed with Newcastle disease virus (titer 10). 6.6 TCID 50The Newcastle disease virus (LaSota strain) was mixed at a volume ratio of 1:1 and then freeze-dried according to the freeze-drying method of Application Example 1. The mixture was then vacuum-sealed and stored at 2-8°C in the dark to obtain the trehalose / Newcastle disease virus freeze-dried preparation.

[0076] II. Test Results 1. Appearance: After freeze-drying, the trehalose / Newcastle disease virus freeze-dried preparation showed slight collapse and the edges of the cake shrank inward; 2. Reconstitution time: Trehalose / Newcastle disease virus lyophilized preparation is reconstituted in water within 1 min 30 s; 3. Moisture content: The moisture content of the trehalose / Newcastle disease virus freeze-dried preparation is 2.9%. 4. Newcastle disease LaSota strain virus titer detection 4.1 Results of Newcastle disease LaSota strain virus titer detection at different stages Newcastle disease LaSota strain original viral titer: 10 6.6 TCID 50 / mL; The titer of lyophilized trehalose / Newcastle disease virus lyophilized formulation: 10 5.5 TCID 50 / mL, titer loss 1.1 log; 37℃ accelerates titer over 7 days: 10 4.5 TCID 50 / mL, with a relative decrease in titer of only 1.0 log after lyophilization; The comparison of Newcastle disease virus (NDV) LaSota strain titers at different stages of NDV stock solution, trehalose / NDV lyophilized formulation, and trehalose / NDV lyophilized formulation after 7 days of accelerated processing at 37°C is shown in the figure below. Figure 10 As shown.

[0077] 5. Results Analysis: Comparative Example 1, using only 5% trehalose as a single protectant for freeze-drying Newcastle disease virus, resulted in slight collapse and edge shrinkage of the freeze-dried cake, poor molding effect, and a reconstitution time of 1 min 30 s; the product moisture content was 2.9%, and the potency of the original solution was 10. 6 TCID 50 / mL, the titer decreased by 1.1 log after lyophilization, and further decreased by 1.0 log after 7 days of accelerated treatment at 37℃, indicating poor viral protection and heat stability. This traditional formulation has mediocre overall performance; as a baseline control, it highlights the technical advantages of the protective agent in this invention.

[0078] Comparative Example 2: BSA protein (0.3%) protectant I. Experimental Methods Formulation: Mix 0.3 g BSA protein, 2.5 g trehalose, 0.08 g magnesium sulfate heptahydrate, and a portion of 0.05 mol / L phosphate buffer (pH 7.0), and bring the volume to 100 mL using the remaining 0.05 mol / L phosphate buffer (pH 7.0). Dissolve by stirring at 4°C, and filter sterilize using a 0.22 μm sterile membrane. This yields a BSA protein (0.3%) protectant. The BSA protein (0.3%) protectant is then mixed with (a titer of 10...) 6.6 TCID 50 The Newcastle disease virus (Newcastle disease strain LaSota) was mixed at a volume ratio of 1:1 and then freeze-dried according to the freeze-drying method of Application Example 1. The mixture was then vacuum-sealed and stored at 2-8°C in the dark to obtain the BSA / Newcastle disease virus freeze-dried preparation.

[0079] II. Test Results 1. Appearance: The BSA / Newcastle disease virus freeze-dried preparation showed slight collapse and the edges of the cake shrank inward; 2. Reconstitution: The reconstitution time for the BSA / Newcastle disease virus lyophilized preparation is 1 min 45 s. After reconstitution, the solution is turbid and has a white precipitate. 3. Moisture content: The moisture content of the BSA / Newcastle disease virus freeze-dried preparation is 3.1%; 4. Newcastle disease LaSota strain virus titer detection 4.1 Results of Newcastle disease LaSota strain virus titer detection at different stages Newcastle disease LaSota strain original viral titer: 10 6.6 TCID 50 / mL; Titer of BSA / Newcastle disease virus lyophilized formulation after freeze-drying: 10 5.8 TCID 50 / mL, titer loss 0.8 log; Accelerated titer at 37℃ for 7 days: 105.2 TCID50 / mL, a decrease of 0.6 log relative to the titer after lyophilization; The comparison of Newcastle disease virus titers at different stages is shown in the figure below, including Newcastle disease virus stock solution, BSA / Newcastle disease virus lyophilized formulation, and BSA / Newcastle disease virus lyophilized formulation after 7 days of accelerated processing at 37°C. Figure 11 As shown.

[0080] 5. BSA protein purity test: The BSA protein purity was 61%, containing starch impurities. To test for starch impurities, an aqueous solution of BSA was added to iodine solution, resulting in a pink color change. The results are shown in the BSA aqueous solution color change graph upon contact with iodine. Figure 12 As shown.

[0081] 6. Results Analysis: After freeze-drying Newcastle disease LaSota virus using a BSA protein (0.3%) protectant prepared with 0.3% BSA + 2.5% trehalose, the reconstituted sample was turbid and showed a white precipitate; the viral log10 TCID... 50 The titer decreased from 6.6 to 5.4, resulting in a 1.2 log loss of activity, indicating limited virus lyophilization protection. Furthermore, the BSA purity was only 61%, and starch impurities were a significant contributing factor to abnormal reconstitution and decreased virus activity. In summary, this protective formulation system exhibited poor protective efficacy and can serve as a control, highlighting the performance advantages of this invention.

[0082] Comparative Example 3: No peptide protectant I. Experimental Methods Formulation: Add 2.5 g of trehalose and 0.08 g of magnesium sulfate heptahydrate sequentially to a portion of 0.05 mol / L phosphate buffer (pH 7.0) at 4°C, and continue stirring at low speed for 10 min at 4°C until completely dissolved. Adjust the volume to 100 mL with the remaining phosphate buffer, then adjust the pH to 7 with phosphate. Filter the solution through a sterile 0.22 μm filter membrane for sterilization, and store in a sealed container at 4°C protected from light to obtain a peptide-free lyophilization protectant. Reconstitute the peptide-free lyophilization protectant with Newcastle disease virus (titer 10). 6.6 TCID 50 The Newcastle disease virus strain (LaSota strain) was mixed at a volume ratio of 1:1 and then freeze-dried according to the freeze-drying method of Application Example 1. The mixture was then vacuum-sealed and stored at 2-8°C in the dark to obtain a peptide-free Newcastle disease virus freeze-dried preparation.

[0083] II. Test Results 1. Appearance: The overall Newcastle disease virus freeze-dried preparation without peptides was slightly collapsed, the surface of the cake was uneven, and the shape was irregular. 2. Reconstitution time: The reconstitution time for the peptide-free Newcastle disease virus lyophilized formulation is 2 min 10 s; 3. Moisture content: The moisture content of the polypeptide-free Newcastle disease virus freeze-dried preparation is 3.8%; 4. Newcastle disease LaSota strain virus titer detection 4.1 Results of Newcastle disease LaSota strain virus titer detection at different stages Newcastle disease LaSota strain original viral titer: 10 6.6 TCID 50 / mL; Titer of the lyophilized Newcastle disease virus preparation without peptides: 10 4.9 TCID 50 / mL, titer loss 1.7log; 37℃ accelerates titer over 7 days: 10 3.8 TCID 50 / mL, the titer decreased by 1.1 log relative to the lyophilized titer; The comparison of Newcastle disease virus titers at different stages, including stock solution, peptide-free lyophilized Newcastle disease virus formulation, and peptide-free lyophilized Newcastle disease virus formulation accelerated at 37°C for 7 days, is shown in the figure below. Figure 13 As shown.

[0084] 5. Results Analysis: Comparative Example 3's formulation did not contain peptide components. After freeze-drying, the product showed slight overall collapse, uneven surface, and poor appearance. Reconstitution took 2 min 10 s, and the moisture content reached 3.8%. The original viral titer was 10. 6 TCID 50 / mL, the titer decreased by 1.7 log after lyophilization, and decreased by another 1.1 log after 7 days of accelerated treatment at 37℃, indicating extremely poor virus protection and heat resistance.

[0085] The peptide-free formulation has obvious defects in overall performance. As a control, it can highlight the synergistic protective advantages brought by the addition of peptides in this invention.

[0086] Comparative Example 4: Blank control without protective agent.

[0087] I. Experimental Methods Newcastle disease virus (titer 10) 6.6 TCID 50 The Newcastle disease virus solution (LaSota strain / mL) was directly freeze-dried according to the freeze-drying method in Application Example 1, without a protectant, to obtain the virus solution freeze-dried agent.

[0088] II. Test Results 1. Appearance: The virus liquid lyophilizer in Comparative Example 4 showed severe collapse and hardened lumps; 2. Resolubility: The resolubility time was 3 min 28 s. The virus liquid lyophilizer in Comparative Example 4 was difficult to resolubilize, and the resolubilized solution was turbid and contained many fragments. 3. Moisture content: The moisture content of the virus lyophilizer in Comparative Example 4 was 4.7%; 4. Newcastle disease LaSota strain virus titer detection Newcastle disease LaSota strain virus titer detection results at different stages Newcastle disease LaSota strain original viral titer: 10 6.6 TCID 50 / mL; Virus fluid lyophilization titer without protectant: 10 4.0 TCID 50 / mL, titer loss 2.6 log; 37℃ accelerates titer over 7 days: 10 2.0 TCID 50 / mL, the titer decreased by 2.0 log relative to the lyophilized titer; The comparison of Newcastle disease virus titers at different stages is shown in the figure below, which includes samples of Newcastle disease virus stock solution, virus lyophilized solution, and virus lyophilized solution after 7 days of accelerated drying at 37°C. Figure 14 As shown.

[0089] 5. Results Analysis: Freeze-dried food without a protective agent lacks skeletal support, resulting in severe collapse and clumping of the freeze-dried cake, substandard appearance, turbidity upon reconstitution, and numerous fragments. The lack of protective components makes it unable to resist freeze-drying damage, with a virus loss titer as high as 2.6 log and extremely poor virus retention activity.

[0090] (II) Comparative Experiment of Freeze-dried Probiotic Lactobacillus plantarum Comparative Example 5: Lyophilized Probiotic Lactobacillus plantarum Experiment with BSA Protectant I. Experimental Methods Formula: Add 0.5 g BSA, 3 g sucrose, 2 g skim milk powder, and 0.1 g sodium ascorbate to a portion of deionized water, then bring the volume to 100 mL with the remaining deionized water, stir to dissolve, and filter with a sterile membrane to obtain BSA protein (0.5%) protectant. Take Lactobacillus plantarum (MRS) and culture to OD 600 =0.7, centrifuged at 4℃ and 6000 rpm for 8 min, and resuspended in PBS to 10 8 The mixture (CFU / mL) was prepared by mixing Lactobacillus plantarum culture and BSA protein (0.5%) protectant at a volume ratio of 1:1, dispensed, and freeze-dried according to the freeze-drying method described in Application Example 1 to obtain the BSA / Lactobacillus plantarum freeze-dried formulation.

[0091] II. Test Results 1. Detection of viable bacteria count during freeze-drying and room temperature storage Plate counts were performed on samples at three key time points: before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C. Actual images of the plate counts for the samples in Comparative Example 5 before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C are shown below. Figure 15 The statistical results of viable bacterial concentration and cell survival rate at each stage are as follows: ① Before freeze-drying: viable count was 3.1 × 10⁻⁶ 8 CFU / mL, with the bacterial survival rate set at 100% (calculation baseline); ② After lyophilization: viable count was 2.39 × 10⁻⁶. 8 CFU / mL, lyophilized bacterial cell survival rate 77.1%; ③ Stored at 25℃ for 6 months: viable bacterial count 1.92×10⁻⁶ 8 The CFU / mL concentration was 61.9% relative to the initial cell survival rate before lyophilization.

[0092] The changes in viable bacterial count and survival rate in Comparative Example 5 before freeze-drying, after freeze-drying, and during storage at 25°C for 6 months are shown in the graph. Figure 16 .

[0093] 2. Reconstitution performance: The reconstitution time is 1 min 12 s. After reconstitution, the BSA / Lactobacillus plantarum lyophilized preparation has a turbid solution with a white precipitate.

[0094] 3. Results Analysis: Comparative Example 5, treated with a BSA-based lyophilization protectant, showed a 77.1% cell survival rate after lyophilization. However, after 6 months of storage at 25°C, the viable cell retention rate was only 61.9%, indicating significant viable cell attenuation. The sample reconstitution was time-consuming, and the reconstituted solution was turbid with a white precipitate, attributed to the precipitation of starch impurities and protein denaturation in the BSA raw material. This system demonstrated poor overall protection for the lyophilized preservation of lactic acid bacteria.

[0095] Comparative Example 6: Lyophilized Experiment of Probiotic Lactobacillus plantarum without Peptide Protectant I. Experimental Methods Formula: Add 3 g of sucrose, 2 g of skim milk powder, and 0.1 g of sodium ascorbate to a portion of deionized water, stir to dissolve, and then use the remaining deionized water to make up to 100 mL. Filter to remove bacteria to obtain a polypeptide-free probiotic protectant. Take Lactobacillus plantarum (MRS) and culture to OD 600 =0.7, centrifuged at 4℃ and 6000 rpm for 8 min, and resuspended in PBS to 10 8 The mixture (CFU / mL) was prepared by mixing Lactobacillus plantarum bacterial culture and peptide-free live probiotic protectant at a volume ratio of 1:1, dispensing, and lyophilizing according to the lyophilization method of Application Example 1 to obtain a peptide-free / Lactobacillus plantarum lyophilized preparation.

[0096] II. Test Results 1. Detection of viable bacteria count during freeze-drying and room temperature storage Plate counts were performed on samples at three key time points: before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C. The resulting images of the plate counts for the samples from Comparative Example 6 before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C are shown below. Figure 17 The statistical results of viable bacterial concentration and cell survival rate at each stage are as follows: ① Before freeze-drying: viable count was 3.1 × 10⁻⁶ 8 CFU / mL, with the bacterial survival rate set at 100% (calculation baseline); ② After lyophilization: viable count was 1.77 × 10⁻⁶. 7 CFU / mL, lyophilized bacterial cell survival rate 5.71%; ③ Stored at 25℃ for 6 months: viable bacterial count 1.14×10⁻⁶. 7CFU / mL, with a relative initial cell survival rate of 3.68% before lyophilization.

[0097] The changes in viable bacterial count and survival rate in Comparative Example 6 before freeze-drying, after freeze-drying, and during storage at 25°C for 6 months are shown in the graph. Figure 18 .

[0098] 2. Reconstitution performance: The reconstitution time of the polypeptide-free / Lactobacillus plantarum lyophilized formulation is 1 min 45 s.

[0099] 3. Results Analysis: The traditional basic protection system relying solely on the combination of sugars and milk powder lacks the anchoring and reinforcement effect of functional peptides on the bacterial cell membrane. It cannot effectively resist freeze-drying dehydration, mechanical damage from ice crystals, and oxidative damage to cells during long-term storage at room temperature. Both the instantaneous survival rate after freeze-drying and the level of live bacteria retention during the subsequent shelf life are seriously insufficient, resulting in a weak overall protection effect.

[0100] Comparative Example 7: Lyophilized Experiment of Probiotic Lactobacillus plantarum without Protectant I. Experimental Methods Bacterial culture: Lactobacillus plantarum, cultured under MRS to OD. 600 =0.7, centrifuged at 4℃ and 6000 rpm for 8 min, and resuspended in PBS to 10 8 CFU / mL; Formula: The above-mentioned Lactobacillus plantarum bacterial solution and sterile water were mixed in a volume ratio of 1:1, dispensed, and then freeze-dried according to the freeze-drying method of Application Example 1 to obtain the Lactobacillus plantarum freeze-dried preparation.

[0101] II. Test Results 1. Detection of viable bacteria count during freeze-drying and room temperature storage Plate counts were performed on samples at three key time points: before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C. The resulting images of the plate counts for the samples in Comparative Example 7 before freeze-drying, after freeze-drying, and after 6 months of sealed storage at 25°C are shown below. Figure 19 The statistical results of viable bacterial concentration and cell survival rate at each stage are as follows: ① Before freeze-drying: viable count was 3.1 × 10⁻⁶ 8 CFU / mL, with the bacterial survival rate set at 100% (calculation baseline); ② After freeze-drying: viable bacterial count was 1.2×10⁶ CFU / mL, and the freeze-dried bacterial survival rate was 0.38%; ③ After storage at room temperature of 25℃ for 6 months: viable bacterial count was 3.72×10⁵ CFU / mL, with a relative initial bacterial survival rate of 0.12% compared to before freeze-drying.

[0102] The changes in viable bacterial count and survival rate in Comparative Example 7 before freeze-drying, after freeze-drying, and during storage at 25°C for 6 months are shown in the graph. Figure 20 .

[0103] 2. Reconstitution: Reconstitution time 2 min 33 s 3. Results Analysis: In the absence of any freeze-drying protectant, *Lactobacillus plantarum* could not withstand the extreme stress and damage of the freeze-drying process, resulting in an extremely low survival rate. During subsequent storage at room temperature, the viable bacteria continued to die rapidly. This blank control fully verifies that a freeze-drying protection system is an essential condition for preserving the activity of lactic acid bacteria, and also highlights the necessity and core technological value of the peptide-protected formulation of this invention.

[0104] (III) Comparative Example of Freeze-Drying Experiment of Industrial Enzyme (α-Amylase) Comparative Example 8: BSA protectant for lyophilization of industrial enzyme (α-amylase) I. Experimental Methods Protection system: 10 mL of α-amylase (1000 U / mL) + 10 mL of BSA solution (1%). The final protection system contains 0.5% BSA and 500 U / mL of α-amylase. Freeze-drying: The protective system was dispensed and freeze-dried according to the freeze-drying method of Application Example 1 to obtain the BSA / α-amylase freeze-dried formulation.

[0105] II. Test Results 1. The protective effect of freeze-drying process on α-amylase activity (1) The enzyme activity test results after lyophilization were as follows: the initial enzyme activity (control before lyophilization) and the enzyme activity (U / ml) of BSA protectant were 500 and 386, respectively. The retention rate of BSA protectant relative to the initial enzyme activity (100% baseline) was 77.2%.

[0106] A biaxial composite plot was plotted using the detection data to obtain a comparison of the changes in enzyme activity and relative activity retention rate of the BSA / α-amylase lyophilized formulations in Comparative Example 8, as shown in the figure below. Figure 21 As shown, this visually reflects the differences in enzyme activity values ​​and activity retention among the groups.

[0107] (2) Heat resistance test at 60℃ 1) Results of enzyme activity detection in the heat resistance test The enzyme activity after heat resistance was 195 U / mL. Based on the enzyme activity after lyophilization, the enzyme activity retention rate of the BSA / α-amylase lyophilized preparation after heat resistance at 60℃ for 30 min was 50.5%. 2) Comparison of enzyme activity between heat-resistant and non-heat-resistant groups A side-by-side bar chart of enzyme activity before lyophilization, after lyophilization, and after heat resistance at 60℃ for 30 min was plotted. This yielded a comparative graph of the heat resistance activities of α-amylase before lyophilization, the lyophilized BSA / α-amylase preparation after lyophilization, and the lyophilized BSA / α-amylase preparation after heat resistance at 60℃ for 30 min in Comparative Example 8. Figure 22The images provide a direct comparison of the heat resistance of the samples.

[0108] 3. Results Analysis: The results above show that when BSA was used as the lyophilization protectant, the enzyme activity retention rate of α-amylase after lyophilization was only 77.2%. After high-temperature treatment at 60℃ for 30 min, the enzyme activity further decreased to 50.52% of the lyophilized level, indicating weak thermal stability. Compared with the tardigrade polypeptide protection system of this invention, BSA has a significant gap in lyophilization protection and heat resistance enhancement effects on α-amylase.

[0109] Comparative Example 9: Lyophilized Industrial Enzyme (α-Amylase) Without Peptide Protectant I. Experimental Methods A system with an α-amylase concentration of 500 U / mL was obtained by using 10 mL of α-amylase (1000 U / mL) + 10 mL of pure water. The system was dispensed and lyophilized according to the lyophilization method of Application Example 1 to obtain an α-amylase lyophilized formulation.

[0110] II. Test Results 1. The protective effect of freeze-drying process on α-amylase activity 1.1 The enzyme activity test results after lyophilization were as follows: the initial enzyme activity (control before lyophilization) and the enzyme activity without peptide protectant (U / ml) were 500 and 157, respectively. The retention rate of enzyme activity without peptide protectant relative to the initial enzyme activity (100% baseline) was 31.4%.

[0111] A biaxial composite plot was created using the detection data to obtain a comparison chart of changes in enzyme activity and relative activity retention rate of the α-amylase lyophilized formulation, as shown below. Figure 23 As shown, this visually reflects the differences in enzyme activity values ​​and activity retention among the groups.

[0112] 2.60℃ heat resistance test 2.1 Results of enzyme activity detection in the heat resistance test The lyophilized α-amylase preparation sample of Comparative Example 9 was subjected to heat treatment at 60℃ for 30 min. The residual enzyme activity after heat treatment was measured and the activity retention rate was calculated, with the enzyme activity after heat treatment as the baseline. The results showed that the enzyme activity after heat treatment was 39 U / mL, and the activity retention rate of the lyophilized α-amylase preparation sample of Comparative Example 9 was 31.2%. 2.2 Comparison of enzyme activity between heat-resistant and non-heat-resistant groups A side-by-side bar chart of enzyme activity before lyophilization, after lyophilization, and after heat resistance at 60℃ for 30 min was plotted. This yielded a comparative chart of the heat resistance activities of α-amylase before lyophilization, the lyophilized α-amylase preparation after lyophilization, and the lyophilized α-amylase preparation after heat resistance at 60℃ for 30 min in Comparative Example 9, as shown below. Figure 24 As shown, the heat resistance of the samples can be compared visually.

[0113] 3. Results Analysis: Comparative Example 9 served as a blank control group without the addition of any lyophilization protective peptides; α-amylase was lyophilized using only a pure water system. After lyophilization, the enzyme activity retained only 31.4% of the initial activity, indicating extremely severe activity loss. Subsequent high-temperature stress at 60℃ for 30 min further reduced the activity, leaving only 31.2% of the lyophilized activity.

[0114] The results demonstrate that the simple freeze-drying process itself can severely damage the structure of α-amylase. Without a dedicated protective component, the enzyme preparation cannot maintain its effective activity, which fully reflects the necessity and outstanding advantages of the tardigrade polypeptide protection system of this invention.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting polypeptides from tardigrades, characterized in that, Includes the following steps: The tardigrades were washed, centrifuged, and frozen sequentially to obtain frozen tardigrades. The frozen insect bodies and pre-cooled phosphate buffer were mixed and then subjected to homogenization, extraction and centrifugation in sequence to obtain crude polypeptide extract. Under stirring conditions, ammonium sulfate solution was added dropwise to the crude polypeptide extract, and the mixture was then subjected to low-temperature standing and low-temperature centrifugation to obtain a refined extract. The refined extract was subjected to ultrafiltration and dialysis sequentially to obtain a purified polypeptide solution. The purified polypeptide solution was subjected to low-temperature vacuum concentration and vacuum freeze-drying / vacuum drying in sequence to obtain tardigrade polypeptide.

2. The extraction method according to claim 1, characterized in that, The freezing temperature is -25℃ to -18℃, and the freezing time is 18 to 45 minutes.

3. The extraction method according to claim 1, characterized in that, The temperature of the pre-cooled phosphate buffer is 3~5℃, the concentration of the pre-cooled phosphate buffer is 0.05 mol / L, the pH of the pre-cooled phosphate buffer is 6.5, and the pre-cooled phosphate buffer contains 0.05~0.2 mmol / L PMSF protease inhibitor.

4. The extraction method according to claim 1, characterized in that, The temperature of the homogenized slurry is 3~5℃, the rotation speed of the homogenized slurry is 7500~13000 rpm, and the homogenization time is 2~6 min.

5. The extraction method according to claim 1, characterized in that, The ammonium sulfate solution is a saturated ammonium sulfate solution; the ammonium sulfate solution is added dropwise until the saturation of ammonium sulfate in the system is 25-35%.

6. The extraction method according to claim 1, characterized in that, The conditions for vacuum freeze drying include: first, pre-freeze drying at -45~-35℃ for 1~3h, then sublimation at -35~-28℃ and a vacuum of 10 Pa for 4~8h, followed by desorption drying at 20~28℃ and a vacuum of 10 Pa for 3~5h.

7. The application of a tardigrade polypeptide extracted by the extraction method according to claims 1 to 6 in a freeze-drying protectant.

8. The application according to claim 7, characterized in that, The freeze-drying protectant is used for freeze-drying stability of live vaccines, probiotics, enzyme preparations, and diagnostic reagents.

9. The application according to claim 8, characterized in that, When the lyophilization protectant is used for the lyophilization stabilization of live vaccines, the lyophilization protectant is prepared from the following raw materials: tardigrade polypeptide, trehalose, magnesium sulfate heptahydrate, and phosphate buffer.

10. The application according to claim 8, characterized in that, When the freeze-drying protectant is used for the freeze-drying stability of probiotics, the freeze-drying protectant is prepared from the following raw materials: tardigrade polypeptide, sucrose, skim milk powder, sodium ascorbate, and deionized water.

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