Giant salamander peptide as well as preparation method and application thereof

Abstract

The invention relates to giant salamander peptide and a preparation method and application thereof, the giant salamander peptide can be obtained by hydrolyzing collagen by neutral protease, pepsin, alkaline protease or trypsin, the average molecular weight is 200-5000 Da, and the polydispersity index is 1.2-1.5. The preparation method comprises the following steps: raw material treatment: obtaining giant salamander skin tissues, degreasing and decolorizing the giant salamander skin tissues with ethanol and deionized water, and air-drying; preliminary enzymolysis and separation: performing preliminary hydrolysis by using pepsin, adjusting the pH value, performing salting-out precipitation, and performing centrifugal collection; purification and preparation of a collagen solution: dialyzing to remove impurities and freeze-drying to prepare collagen, and dissolving to form the collagen solution; enzymolysis, separation and purification: adding protease for hydrolysis, centrifuging after enzyme deactivation, filtering, carrying out molecular weight grading purification, and freeze-drying to obtain the giant salamander peptide. The invention provides the method for efficiently extracting the giant salamander collagen peptide, the purity and uniformity of the giant salamander peptide are improved, the dispersity index is reduced, and the method is suitable for the fields of functional health care products, medicines and cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a giant salamander peptide and a preparation method and application thereof. Background Art

[0002] Due to its unique biological activity, giant salamander peptides have a wide range of application value in functional foods, medicines and cosmetics. As a protein hydrolysate, giant salamander peptides are rich in a variety of small molecule active peptides, which can promote biological effects such as cell repair, anti-oxidation, and immune regulation. They are particularly suitable for anti-aging, skin repair and sports nutrition supplements. However, in practical applications, the performance requirements of giant salamander peptides are mainly concentrated on the uniformity of its molecular weight distribution, the stability of biological activity and solubility. First, in order to ensure the efficient absorption of giant salamander peptides in the body, its molecular weight usually needs to be controlled between 200 and 5000 Da to balance the absorption rate and biological effect. Secondly, the biological activity of peptides depends on their structural characteristics, and the control of polydispersity index (PDI) is crucial to ensure the consistency between product batches. Lower PDI helps to improve the uniformity and stability of the product. In addition, the solubility of giant salamander peptides in different pH and temperature environments directly affects its application range, especially in the fields of medicine and functional foods. Good water solubility can improve its bioavailability. Therefore, optimizing the preparation process of giant salamander peptides so that they have a controllable molecular weight range, low dispersity index and excellent solubility properties is of great significance for improving product quality, expanding market applications and promoting technological progress in the industry.

[0003] At present, there are still many challenges in the preparation technology of giant salamander peptides, especially in terms of the diversity control of peptide types and the uniformity of molecular weight distribution. There is still a lot of room for optimization. For example, the Chinese patent with publication number CN113637717B discloses a giant salamander oligopeptide and its preparation method and application, but the method has low selectivity for different types of proteases during the enzymatic hydrolysis process, resulting in a single type of giant salamander peptides obtained, limiting the diversity of its biological activity. In addition, the method failed to effectively control the polydispersity index during the peptide separation and purification process, resulting in a wide molecular weight distribution of the obtained giant salamander peptides, affecting the uniformity and stability of the product, and thus may affect its functional performance in different application fields. This is mainly due to the lack of control of the selection of proteases and enzymatic conditions, as well as the lack of optimization of subsequent separation and purification technologies, such as the selection of molecular weight cutoff membranes and the precise control of dialysis conditions, which still need further improvement. Therefore, in view of the problems of single peptide types and high dispersity index in the preparation of giant salamander peptides, it is urgent to develop an optimized enzymatic hydrolysis and separation and purification strategy to obtain a more uniform, highly active giant salamander peptide product suitable for a variety of application scenarios. Summary of the invention

[0004] (1) Technical issues solved The purpose of the present invention is to provide a giant salamander peptide and a preparation method and application thereof, so as to solve the problem that the giant salamander peptide currently has a single type and a high dispersion index.

[0005] (2) Technical solution In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing a giant salamander peptide comprises the following steps: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use.

[0006] S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue is enzymatically hydrolyzed with a pepsin solution, and the pH value is adjusted to complete the preliminary digestion; the enzymatic hydrolyzate is centrifuged to collect the supernatant, a salt solution is added for salting out, and the precipitate is collected by centrifugation.

[0007] S3. Purification and preparation of collagen solution: dialyze the precipitate to remove impurities, and freeze-dry to obtain a crude collagen extract; dissolve the crude extract in an acidic solution to prepare a collagen solution, and stir until it is uniform and transparent.

[0008] S4. Enzymatic hydrolysis and separation and purification: proteases are added to the collagen solution for enzymatic hydrolysis, and the enzymes are inactivated after completion; the enzymatic hydrolysis products are centrifuged and filtered, separated and purified using a molecular weight cutting membrane, and freeze-dried to obtain giant salamander peptide powder.

[0009] Furthermore, the detailed process of the S1 raw material processing is: using healthy farmed giant salamanders as raw materials, selecting individuals with an average weight of 4~6 kg and a body length of 70~75 cm, placing the giant salamanders in an environment of 12~16°C, anesthetizing them with a MS-222 solution with a concentration of 0.5~0.7 g / L, dissecting them, obtaining skin tissue, and cutting the skin tissue into small pieces of about 4mm×4mm~6mm×6mm, then placing the obtained small pieces in a solvent mixed with ethanol and deionized water in a volume ratio of (1.0~1.5):1, soaking them at 20~30°C for 10~14h until fat and impurities are removed, after soaking, repeatedly washing the skin tissue with deionized water until no obvious color flows out, and placing the washed skin tissue in an environment of 20~25°C to air dry for 24~36h for use.

[0010] Furthermore, the detailed process of the preliminary enzymatic hydrolysis and separation of S2 is as follows: the air-dried skin tissue is placed in a 0.1~0.3% mass fraction pepsin solution, the pH of the solution is adjusted to 1.8~2.2, stirring is maintained at 35~40°C, the enzymatic hydrolysis reaction time is 4~6h, after the enzymatic hydrolysis is completed, the pH is adjusted to 7.5~8.5 with 0.4~0.6mol / L acetic acid solution, and then adjusted to 1.8~2.2, and allowed to stand for 10~20min to ensure complete digestion, the enzymatic hydrolysis solution is centrifuged at a speed of 7000~9000 r / min for 10~20min, the supernatant is collected, and 4~6 mol / L NaCl solution is slowly added to the supernatant, the supernatant and the NaCl solution are mixed at a molar ratio of (0.8~1.2):1, stirred to form a flocculent precipitate, and then centrifuged again at a speed of 7000~9000r / min for 10~20min, and the resulting precipitate is collected for later use.

[0011] Furthermore, the detailed process of the S3 purification and collagen solution preparation is as follows: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 8,000 to 14,000 Da, deionized water is used as the dialysis medium, and dialyzed for 36 to 48 hours at 2 to 6 ° C, and the dialysate is replaced every 4 to 6 hours until the impurities are completely removed. After the dialysis is completed, the dialysate is transferred to a freeze dryer and freeze-dried at -45 to -55 ° C to obtain a crude collagen extract. The freeze-dried crude extract is dissolved in a 0.4 to 0.6 mol / L acetic acid solution to prepare a collagen solution with a concentration of 0.8 to 1.2%, and stirred at a stirring rate of 250 to 350 r / min for 20 to 40 minutes until the solution is uniform and transparent to obtain a collagen solution for standby use.

[0012] Further, the detailed process of the S4 enzymatic hydrolysis and separation and purification is as follows: adding protease to the collagen solution for enzymatic hydrolysis, after the enzymatic hydrolysis is completed, placing the reaction solution in a 90-100°C water bath and heating for 8-12 minutes to inactivate the enzyme activity, centrifuging the enzymatic hydrolyzate after enzyme inactivation at a speed of 7000-9000 r / min for 10-20 minutes, collecting the supernatant, and filtering through a 0.45 μm filter membrane, separating and purifying the filtered solution by cutting the membrane, collecting collagen peptide solutions of different molecular weight ranges, diluting each collagen peptide solution of the molecular weight range to an appropriate concentration, transferring to a freeze dryer, and freeze drying at -45--55°C to obtain giant salamander peptide powders of different molecular weights; The present invention adopts a systematic enzymatic hydrolysis and separation and purification design, which is mainly used to enhance the purity, uniformity and biological activity of giant salamander peptides. Through scientific and reasonable raw material processing, precisely controlled preliminary enzymatic hydrolysis, efficient impurity removal and optimized molecular weight fractionation purification, a high degree of synergy is formed between each step, thereby improving the quality and functional characteristics of the target product. In the raw material processing stage, a mixed solvent of ethanol and deionized water is used for degreasing and decolorization, effectively removing interfering components such as lipids and pigments, while maintaining the natural structure of collagen, providing high-quality raw materials for subsequent enzymatic hydrolysis. Subsequently, in the preliminary enzymatic hydrolysis process, pepsin is used to selectively hydrolyze the giant salamander skin tissue under specific pH and temperature conditions, so that collagen is moderately degraded into soluble proteins, and the target protein is further concentrated by combining salting-out precipitation to ensure the stability of the enzymatic hydrolysis product. During the purification process, small molecule impurities and inorganic salts are effectively removed by dialysis technology, the purity of collagen is improved, and its structural integrity is maintained by freeze-drying technology, creating favorable conditions for subsequent precise enzymatic hydrolysis. In the final enzymatic hydrolysis and separation and purification stage, specific proteases are added to the collagen solution to achieve deep degradation of collagen. At the same time, by strictly controlling the enzymatic hydrolysis conditions, the diversity of species and controllability of the molecular weight distribution of the final giant salamander peptides are ensured. After the enzyme is inactivated, the enzymatic hydrolysis products are subjected to centrifugation, filtration and molecular weight cutting membrane separation in sequence, so that giant salamander peptides of different molecular weight levels can be efficiently separated, and finally a stable peptide powder is obtained by freeze-drying. The entire process ensures the uniform molecular weight distribution of giant salamander peptides, reduces the polydispersity index, and avoids the loss of activity caused by excessive degradation through precise pH adjustment, temperature control and purification methods, thereby enhancing its application value. This method not only optimizes the synergy of enzymatic hydrolysis and purification, improves the extraction efficiency of giant salamander peptides, but also significantly improves the uniformity and solubility of the final product, so that it can better meet the application needs in the fields of functional foods, medicines and cosmetics.

[0013] Furthermore, the protease is any one of neutral protease, pepsin, alkaline protease and trypsin; When the protease is a neutral protease, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 48~52℃, pH value 6.8~7.2, enzyme dosage 5500~6500 U / g, solid-liquid ratio 0.08~0.12g / mL; When the protease is pepsin, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 35~39℃, pH value 1.8~2.2, enzyme dosage 5500~6500 U / g, solid-liquid ratio 0.08~0.12g / mL; When the protease is alkaline protease, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3-5 hours, temperature 48-52 °C, pH value 7.8-8.2, enzyme dosage 5500-6500 U / g, solid-liquid ratio 0.08-0.12 g / mL; When the protease is trypsin, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 38~42℃, pH value 7.8~8.2, enzyme dosage 5500~6500 U / g, and solid-liquid ratio 0.08~0.12g / mL.

[0014] The present invention adopts a design of precise enzymatic hydrolysis control, which is mainly used to enhance the molecular weight uniformity, purity and biological activity of giant salamander peptides to meet the application requirements in the fields of functional foods, medicines and cosmetics. By selecting any one of neutral proteases, pepsin, alkaline proteases or trypsin, and acting on collagen under strictly controlled enzymatic hydrolysis conditions, the directional degradation of giant salamander peptides is achieved. According to the optimal pH, temperature and enzyme dosage of different proteases, the enzymatic hydrolysis time and liquid-to-solid ratio are optimized respectively to ensure the controllability of protein degradation while retaining the integrity of functional peptide segments. Pepsin can efficiently hydrolyze specific peptide bonds in an acidic environment and promote the initial depolymerization of collagen, while alkaline proteases and trypsin further act under higher pH conditions to break the peptide chains to the target molecular weight range, thereby obtaining a more uniform giant salamander peptide product. In addition, the action of neutral proteases under conditions close to physiological pH can reduce the damage to the amino acid side chains and enhance the functional stability of the final product. The synergistic effect between the various enzymatic hydrolysis steps not only helps to improve the efficiency of enzymatic hydrolysis, but also reduces nonspecific hydrolysis and polydispersity index by optimizing reaction conditions, ensuring the acquisition of high-purity giant salamander peptides with controllable molecular weight distribution. This optimized enzymatic hydrolysis strategy effectively avoids the loss of activity caused by excessive degradation, while improving the solubility and absorption utilization of the target product, making it better adapted to a variety of application scenarios, and ultimately enhancing the industrial production value of giant salamander peptides.

[0015] Furthermore, the method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 450-600 parts by weight of benzene are added to a reactor, 1.0-2.0 parts of anionic initiator sec-butyl lithium are added to the reactor via a syringe in a glove box, 50-80 parts of isoprene are added to the reactor, and the reaction is continued for 8-12 hours, and then 100-150 parts of styrene are added to the reactor via a syringe in the glove box, and the polymerization is carried out for 36-40 hours, and then benzene is removed from the reactor, and diphenylethylene is added in an amount of 10 times the molar amount of sec-butyl lithium, 500-600 mL of tetrahydrofuran is directly distilled into the reactor, and then the reactor is cooled to -60--78°C, and then 30-50 parts of 4-vinylpyridine are added, and the reaction is continued for 60-90 minutes, followed by 5-10 The triblock copolymer was terminated by adding parts of methanol, and the triblock copolymer was dissolved in chloroform, and then added dropwise into excess methanol for two precipitation purifications. The precipitate was collected by filtration and washed with methanol three times. Finally, the purified copolymer was placed in a vacuum drying oven and dried at 40-50°C for 24-48 h until the solvent was completely removed, thereby finally obtaining an isoprene-styrene-4-vinylpyridine (ISV) block copolymer.

[0016] A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1: (4.0~6.0), the concentration of the glycerol solution was 8.0~12.0 wt.%, and the solvent was tetrahydrofuran and 1,4-dioxane in a mass ratio of 4: (6.0~8.0). All raw materials were placed in a stirring device with a stirring rate of 350~450rpm for 60~80min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 12~14h to ensure that the glycerol and block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer, and the liquid film thickness was adjusted to 220~250μm. The coated substrate was quickly transferred to a deionized water precipitation bath, and the precipitation bath temperature was maintained at 22~25 ℃, soak for 12~15min to remove the solvent and glycerol and form a complete mesoporous membrane structure. Subsequently, the membrane is taken out from the deionized water and rinsed with deionized water 3~4 times, each rinse time is 8~12min, to completely remove the residual solvent and glycerol, and finally obtain the mesoporous membrane.

[0017] A3: Regulation of membrane pore size: Soak the mesoporous membrane in a citric acid-sodium citrate buffer solution with a pH of 3.2-3.5 for 20-25 min. The stirring rate of the buffer solution is 180-220 rpm and the soaking time is 60-90 min. Then, rinse the cut membrane with deionized water for 4-5 times and place the membrane in a vacuum drying oven at 50-55 °C for 16-20 h until the membrane is completely dry to obtain the cut membrane.

[0018] The present invention adopts the design of cutting membrane mainly to reduce the dispersibility index, so as to improve the molecular weight uniformity and purity of giant salamander peptide, thereby optimizing its biological activity and application performance. First, through the synthesis of isoprene-styrene-4-vinylpyridine block copolymer, sec-butyl lithium is used to initiate the polymerization reaction of isoprene and styrene, ensuring that the copolymer has good compatibility and controllable microstructure, and the polymerization is terminated by diphenylethylene to improve the stability of the polymer. Subsequently, tetrahydrofuran and 1,4-dioxane system are used to dissolve the copolymer, and mixed with glycerol solution to form a stable casting solution, and a uniform mesoporous membrane structure is achieved under stirring and precipitation bath treatment. The formation of the mesoporous membrane depends on the phase separation effect of glycerol, so that a regular pore structure is formed in the membrane, thereby giving the cutting membrane excellent molecular sieving characteristics. In the process of membrane pore size regulation, the swelling behavior of the membrane material is regulated by the acidic environment of citric acid-sodium citrate buffer solution, so that the pore size reaches a suitable separation range, and the uniformity of the membrane pore size is optimized by controlling the immersion time and stirring rate. Finally, the residues were removed by washing with deionized water, and the membrane structure was stabilized under vacuum drying conditions to obtain an efficient cutting membrane. The entire process flow, through the synergistic effect of copolymer preparation, mesoporous membrane formation and pore size regulation, enables the cutting membrane to have high selectivity and stability in the molecular weight screening process of giant salamander peptides, thereby effectively reducing the dispersion index, improving the uniformity and purity of the final product, and ensuring its wide application value in functional foods, medicines and cosmetics.

[0019] The present invention also provides a giant salamander peptide, which is prepared by the above method; The giant salamander peptide is any one of neutral protease collagen peptide, pepsin collagen peptide, alkaline protease collagen peptide or trypsin collagen peptide; The average molecular weight of the giant salamander peptide ranges from 200 to 5000Da; The polydispersity index of the giant salamander peptide is 1.2-1.5.

[0020] The invention also discloses the application of giant salamander peptide in the fields of functional health products, medicine and functional cosmetics.

[0021] The present invention adopts the design of precise enzymolysis and separation and purification, which is mainly used to prepare giant salamander peptides with uniform molecular weight, high purity and excellent biological activity, so as to meet the application requirements in the fields of functional health products, medicine and functional cosmetics. By optimizing the enzymolysis process, neutral protease, pepsin, alkaline protease or trypsin are used to carry out directional degradation of collagen, so as to ensure that the molecular weight of the obtained giant salamander peptide is controlled within the range of 200~5000Da, thereby taking into account both biological activity and absorption efficiency. At the same time, by strictly regulating the enzymolysis conditions, including pH, temperature, enzyme dosage and enzymolysis time, the polydispersity index of the giant salamander peptide is stabilized at 1.2~1.5, ensuring the uniformity of its molecular weight distribution and improving the stability between product batches. The synergistic effect of various proteases under specific conditions enables the obtained giant salamander peptide to reach an optimized state in terms of structural integrity, bioavailability and solubility. In addition, combined with efficient separation and purification technology, including centrifugation, filtration and molecular weight cutting membrane screening, non-target products and impurities are further removed to improve the quality of the final product. The resulting giant salamander peptide can be used to enhance physiological regulation in the field of functional health products, has potential applications in the field of medicine such as promoting tissue repair and anti-inflammatory, and can provide moisturizing, anti-aging and skin barrier repair in the field of functional cosmetics. The entire process system achieves the preparation of high-quality giant salamander peptides through precise enzymatic hydrolysis control, optimized separation and purification strategies, and strict molecular weight control, laying a solid foundation for its promotion in multiple high-end application fields.

[0022] (3) Beneficial technical effects 1. The present invention achieves high purity and low dispersity index of giant salamander peptides by optimizing enzymatic hydrolysis and separation and purification processes, which improves extraction efficiency, reduces impurity residues, and ensures product batch stability and biological activity compared to the prior art. Ethanol and deionized water are used for degreasing and decolorization to keep the collagen structure intact, pepsin is used for preliminary hydrolysis combined with salting out and concentration to improve enzymatic hydrolysis efficiency, dialysis is used to remove small molecule impurities and freeze-drying stabilizes collagen, and then specific proteases are added in sequence for fine degradation, combined with molecular weight cutting membranes for precise screening, effectively controlling the molecular weight range, reducing the polydispersity index, and each step is synergistically optimized to ensure the quality of the final product, which is widely applicable to the fields of functional foods, medicines and cosmetics.

[0023] 2. The present invention improves the molecular weight uniformity, purity and biological activity of giant salamander peptides through precise enzymatic hydrolysis control and step-by-step optimization, which is significantly better than the existing technology. According to the characteristics of different proteases, the pH, temperature and enzyme dosage are optimized to achieve directional degradation, avoid nonspecific hydrolysis, and improve the stability of the target peptide segment. Pepsin is used for preliminary depolymerization, alkaline protease and trypsin further refine the molecular weight, and neutral protease maintains the activity integrity. The synergistic effect of each enzyme improves the enzymatic hydrolysis efficiency and reduces the polydispersity index, ensuring the functional stability and solubility of the final product, and is widely used in the fields of functional food, medicine and cosmetics.

[0024] 3. The present invention achieves precise screening of the molecular weight of giant salamander peptides by optimizing the preparation and regulation of the cutting membrane. Compared with the prior art, it improves the molecular weight uniformity and reduces the dispersity index, thereby enhancing its biological activity and application performance. Isoprene-styrene-4-vinylpyridine block copolymer ensures the stability of the membrane material, glycerol induces the formation of a uniform mesoporous structure, and citric acid-sodium citrate buffer solution accurately controls the membrane pore size. The synergistic effect of each step enables the cutting membrane to have efficient screening capabilities. The final product has broad application prospects in the fields of functional foods, medicines and cosmetics, improves industry standards, and promotes the development of high-end peptide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the external Fourier spectrum of the giant salamander peptide prepared in Example 1 of the present invention.

[0026] Figure 2 This is a microscopic morphology of the cutting film prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0028] Example 1

[0029] A method for preparing a giant salamander peptide comprises the following steps: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use. The detailed process is: Use healthy farmed giant salamanders as raw materials, select individuals with an average weight of 4kg and a body length of 70cm, place the giant salamanders in a 12℃ environment, anesthetize them with a 0.5 g / L MS-222 solution, dissect them, obtain skin tissue, cut the skin tissue into small pieces of about 4mm × 4mm, and then place the small pieces in a solvent mixed with ethanol and deionized water at a volume ratio of 1.0:1, soak them at 20℃ for 10h until fat and impurities are removed. After soaking, wash the skin tissue repeatedly with deionized water until no obvious color flows out, and place the washed skin tissue at 20℃ to air-dry for 24h for later use.

[0030] S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue was enzymatically hydrolyzed with pepsin solution, and the pH value was adjusted to complete the preliminary digestion; the enzymatic hydrolyzate was centrifuged to collect the supernatant, and a salt solution was added for salting out, and the precipitate was collected by centrifugation. The detailed process is as follows: the air-dried skin tissue was placed in a 0.1% mass fraction pepsin solution, the pH of the solution was adjusted to 1.8, and stirring was maintained at 35 °C. The enzymatic hydrolysis reaction time was 4h. After the enzymatic hydrolysis was completed, the pH was adjusted to 7.5 with 0.4 mol / L acetic acid solution, and then adjusted to 1.8, and allowed to stand for 10min to ensure complete digestion. The enzymatic hydrolyzed solution was centrifuged at 7000 r / min for 10min, the supernatant was collected, and 4 mol / L NaCl solution was slowly added to the supernatant. The supernatant and NaCl solution were mixed at a molar ratio of 0.8:1, stirred until a flocculent precipitate was formed, and then centrifuged again at 7000 r / min for 10min, and the resulting precipitate was collected for later use.

[0031] S3. Purification and preparation of collagen solution: The precipitate is dialyzed to remove impurities, and lyophilized to obtain a crude collagen extract; the crude extract is dissolved in an acidic solution to prepare a collagen solution, and stirred until it is uniform and transparent. The detailed process is as follows: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 8000Da, deionized water is used as the dialysis medium, and the dialysis is performed at 2°C for 36 hours, and the dialysis fluid is replaced every 4 hours until the impurities are completely removed. After the dialysis is completed, the dialysis fluid is transferred to a freeze dryer and lyophilized at -45°C to obtain a crude collagen extract. The lyophilized crude extract is dissolved in a 0.4mol / L acetic acid solution to prepare a collagen solution with a concentration of 0.8%, and stirred at a stirring rate of 250r / min for 20 minutes until the solution is uniform and transparent, and the collagen solution is obtained for standby use.

[0032] S4. Enzymatic hydrolysis and separation and purification: Add proteases to the collagen solution for enzymatic hydrolysis, and inactivate the enzymes after completion; centrifuge and filter the enzymatic hydrolysis products, separate and purify them using a molecular weight cutting membrane, and freeze-dry to obtain giant salamander peptide powder. The detailed process is: add neutral protease to the collagen solution for enzymatic hydrolysis, and the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3h, temperature 48℃, pH value 6.8, enzyme dosage 5500U / g, and solid-liquid ratio 0.08g / mL; after the enzymatic hydrolysis is completed, place the reaction solution in a 90℃ water bath and heat for 8min to inactivate the enzyme activity. The enzymatic hydrolysis products after enzyme inactivation are centrifuged at a speed of 7000r / min for 10min, collect the supernatant, and filter through a 0.45μm filter membrane. The filtered solution is separated and purified by a cutting membrane, and collagen peptide solutions of different molecular weight ranges are collected respectively. The collagen peptide solutions of each molecular weight range are diluted to an appropriate concentration, transferred to a freeze dryer, and freeze-dried at -45℃ to obtain giant salamander peptide powders of different molecular weights; The method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 450 parts by weight of benzene were added to a reactor, 1.0 part of anionic initiator sec-butyl lithium was added to the reactor via a syringe in a glove box, 50 parts of isoprene were added to the reactor, and the reaction was continued for 8 hours, then 100 parts of styrene were added to the reactor via a syringe in a glove box, and the polymerization was continued for 36 hours, then benzene was removed from the reactor, and diphenylethylene was added in an amount 10 times the molar amount of sec-butyl lithium, and 500 parts of isoprene were added to the reactor. mL of tetrahydrofuran was directly distilled into the reactor, and then the reactor was cooled to -60°C, and then 30 parts of 4-vinylpyridine were added, and the reaction was continued for 60 minutes. Then, 5 parts of methanol were used to terminate the triblock copolymer, and the triblock copolymer was dissolved in chloroform, and then added dropwise to excess methanol for two precipitation purifications. The precipitate was collected by filtration and washed with methanol 3 times. Finally, the purified copolymer was placed in a vacuum drying oven and dried at 40°C for 24 hours until the solvent was completely removed, and finally isoprene-styrene-4-vinylpyridine (ISV) block copolymer was obtained.

[0033] A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1:4.0, the concentration of the glycerol solution was 8.0 wt.%, and the solvent was tetrahydrofuran and 1,4-dioxane in a mass ratio of 4:6.0. All raw materials were placed in a stirring device with a stirring rate of 350 rpm and stirred for 60 min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 12 h to ensure that the glycerol and the block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer, and the liquid film thickness was adjusted to 220 μm. The coated substrate was quickly transferred to a deionized water precipitation bath, and the precipitation bath temperature was maintained at 22 °C. It was soaked for 12 min to remove the solvent and glycerol to form a complete mesoporous membrane structure. Subsequently, the membrane was taken out of the deionized water and rinsed with deionized water for 3 times, each rinse time was 8 min, to completely remove the residual solvent and glycerol, and finally a mesoporous membrane was obtained.

[0034] A3: Regulation of membrane pore size: The mesoporous membrane was immersed in a citric acid-sodium citrate buffer solution with a pH of 3.2. The stirring rate of the buffer solution was 180 rpm and the immersion time was 60 min. The cut membrane was then rinsed with deionized water for 4 times and placed in a 50 °C vacuum drying oven for 16 h until the membrane was completely dry to obtain the cut membrane.

[0035] The giant salamander peptide is a neutral protease collagen peptide; the average molecular weight of the giant salamander peptide is in the range of 200 Da; and the polydispersity index of the giant salamander peptide is 1.2.

[0036] This embodiment achieves the precise preparation of giant salamander peptides, and adopts optimized enzymatic hydrolysis and separation and purification processes to stabilize the average molecular weight of the obtained giant salamander peptides at 200Da, and reduce the polydispersity index to 1.2, significantly improving the molecular weight uniformity and product batch stability. Through the directional degradation of neutral proteases, combined with the precise screening of molecular weight cutting membranes, the high purity and high biological activity of giant salamander peptides are ensured, while improving their solubility and bioavailability, making them have better application value in the fields of functional foods, medicines and cosmetics, and promoting the industrial production and market application of high-end peptide products.

[0037] Through the Fourier transform infrared spectroscopy (FTIR) analysis in Figure 1, it can be observed that Amide A (3367 cm -1 )、Amide B(2934 cm -1 )、Amide I(1654 cm -1 )、Amide II(1420 cm -1 ) and Amide III (1151cm -1), which proves that the giant salamander peptide (ADFP) extracted in this study has a typical peptide structure, indicating that its chemical composition is in line with expectations. In addition, Figure 2 shows the microscopic morphology of the cut membrane prepared in Example 1, and the uniformly distributed pore structure can be clearly observed, which provides a stable physical basis for the precise molecular weight screening of giant salamander peptides, helps to reduce its dispersity index (PDI), and ensures the uniformity of molecular weight distribution. These results fully verify that the preparation process of the present invention can effectively obtain high-purity and uniform giant salamander peptides, and prove the scientificity and feasibility of the method.

[0038] Example 2

[0039] A method for preparing a giant salamander peptide comprises the following steps: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use. The detailed process is as follows: Use healthy farmed giant salamanders as raw materials, select individuals with an average weight of 4.6 kg and a body length of 71.5 cm, place the giant salamanders in an environment of 13.2 ℃, anesthetize them with a concentration of 0.56 g / L MS-222 solution, dissect them, obtain skin tissue, cut the skin tissue into small pieces of about 4.6 mm × 4.6 mm, and then place the small pieces in a solvent mixed with ethanol and deionized water at a volume ratio of 1.15:1, soak them at 23 ℃ for 11.2 hours until fat and impurities are removed, and after soaking, repeatedly wash the skin tissue with deionized water until no obvious color flows out, and place the washed skin tissue at 21.5 ℃ to air-dry for 27.6 hours for later use.

[0040] S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue was enzymatically hydrolyzed with pepsin solution, and the pH value was adjusted to complete the preliminary digestion; the enzymatic hydrolyzate was centrifuged to collect the supernatant, and a salt solution was added for salting out, and the precipitate was collected by centrifugation. The detailed process is as follows: the air-dried skin tissue was placed in a 0.16% mass fraction pepsin solution, the pH of the solution was adjusted to 1.92, and stirring was maintained at 36.5 °C. The enzymatic hydrolysis reaction time was 4.6 h. After the enzymatic hydrolysis was completed, the pH was adjusted to 7.8 with 0.46 mol / L acetic acid solution, and then adjusted to 1.92, and allowed to stand for 13 min to ensure complete digestion. The enzymatic hydrolyzed solution was centrifuged at 7600 r / min for 13 min, the supernatant was collected, and 4.6 mol / L NaCl solution was slowly added to the supernatant. The supernatant and NaCl solution were mixed at a molar ratio of 0.92:1, stirred until a flocculent precipitate was formed, and then centrifuged again at 7600 r / min for 13 min, and the resulting precipitate was collected for later use.

[0041] S3. Purification and preparation of collagen solution: The precipitate is dialyzed to remove impurities, and lyophilized to obtain a crude collagen extract; the crude extract is dissolved in an acidic solution to prepare a collagen solution, and stirred until it is uniform and transparent. The detailed process is as follows: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 9800 Da, deionized water is used as the dialysis medium, and the dialysis is performed at 3.2 °C for 39.6 hours, and the dialysis fluid is replaced every 4.6 hours until the impurities are completely removed. After the dialysis is completed, the dialysis fluid is transferred to a freeze dryer and lyophilized at -48 °C to obtain a crude collagen extract. The lyophilized crude extract is dissolved in a 0.46 mol / L acetic acid solution to prepare a collagen solution with a concentration of 0.92%, and stirred at a stirring rate of 280r / min for 26 minutes until the solution is uniform and transparent, and the collagen solution is obtained for standby use.

[0042] S4. Enzymatic hydrolysis and separation and purification: proteases were added to the collagen solution for enzymatic hydrolysis, and the enzymes were inactivated after completion; the enzymatic hydrolysis products were centrifuged and filtered, separated and purified using a molecular weight cutting membrane, and freeze-dried to obtain giant salamander peptide powder. The detailed process is: pepsin was added to the collagen solution for enzymatic hydrolysis, and the enzymatic hydrolysis conditions were: enzymatic hydrolysis time 3.6h, temperature 36.2℃, pH value 1.92, enzyme dosage 5800 U / g, and solid-liquid ratio 0.09g / mL; after the enzymatic hydrolysis was completed, the reaction solution was placed in a 93℃ water bath and heated for 9.2min to inactivate the enzyme activity. The enzymatic hydrolysis products after enzyme inactivation were centrifuged at a speed of 7600 r / min for 13min, the supernatant was collected, and filtered through a 0.45μm filter membrane. The filtered solution was separated and purified by a cutting membrane, and collagen peptide solutions of different molecular weight ranges were collected respectively. The collagen peptide solutions of each molecular weight range were diluted to an appropriate concentration, transferred to a freeze dryer, and freeze-dried at -48℃ to obtain giant salamander peptide powders of different molecular weights; The method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 495 parts by weight of benzene were added to a reactor, 1.3 parts of anionic initiator sec-butyl lithium were added to the reactor via a syringe in a glove box, 59 parts of isoprene were added to the reactor, and the reaction was continued for 9.2 hours. Subsequently, 115 parts of styrene were added to the reactor via a syringe in the glove box, and the polymerization was continued for 37.2 hours. Subsequently, benzene was removed from the reactor, and diphenylethylene in an amount 10 times the molar amount of sec-butyl lithium was added. 530 parts of isoprene were added to the reactor, and the reaction was continued for 9.2 hours. mL of tetrahydrofuran was directly distilled into the reactor, and then the reactor was cooled to -65.4°C, and then 36 parts of 4-vinylpyridine were added, and the reaction was continued for 69 minutes. Then, 6.5 parts of methanol were used to terminate the triblock copolymer, and the triblock copolymer was dissolved in chloroform, and then added dropwise to excess methanol for two precipitation purifications. The precipitate was collected by filtration and washed with methanol 3 times. Finally, the purified copolymer was placed in a vacuum drying oven and dried at 43°C for 31.2 hours until the solvent was completely removed, and finally isoprene-styrene-4-vinylpyridine (ISV) block copolymer was obtained.

[0043] A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1:4.6, the concentration of the glycerol solution was 9.2 wt.%, and the solvent was tetrahydrofuran and 1,4-dioxane mixed in a mass ratio of 4:6.6. All raw materials were placed in a stirring device with a stirring rate of 380 rpm and stirred for 66 min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 12.6 h to ensure that the glycerol and the block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer, and the liquid film thickness was adjusted to 229 μm. The coated substrate was quickly transferred to a deionized water precipitation bath, and the precipitation bath temperature was maintained at 22.9 ℃. It was soaked for 12.9 min to remove the solvent and glycerol and form a complete mesoporous membrane structure. Subsequently, the membrane was taken out of the deionized water and rinsed with deionized water for 3 times, each rinse time was 9.2 min, to completely remove the residual solvent and glycerol, and finally a mesoporous membrane was obtained.

[0044] A3: Regulation of membrane pore size: The mesoporous membrane was immersed in a citric acid-sodium citrate buffer solution with a pH of 3.29. The stirring rate of the buffer solution was 192 rpm and the immersion time was 69 min. The cut membrane was then rinsed with deionized water for 4 times and placed in a vacuum drying oven at 51.5 °C for 17.2 h until the membrane was completely dry to obtain the cut membrane.

[0045] The giant salamander peptide is a pepsin collagen peptide; the average molecular weight of the giant salamander peptide ranges from 1640 Da; and the polydispersity index of the giant salamander peptide is 1.29.

[0046] Example 3

[0047] A method for preparing a giant salamander peptide comprises the following steps: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use. The detailed process is: Use healthy farmed giant salamanders as raw materials, select individuals with an average weight of 5.2 kg and a body length of 73 cm, place the giant salamanders in an environment of 14.4 ℃, anesthetize them with a concentration of 0.62 g / L MS-222 solution, dissect them, obtain skin tissue, cut the skin tissue into small pieces of about 5.2 mm × 5.2 mm, and then place the obtained small pieces in a solvent mixed with ethanol and deionized water in a volume ratio of 1.3:1, soak them at 26 ℃ for 12.4 hours until fat and impurities are removed, and after soaking, repeatedly wash the skin tissue with deionized water until no obvious color flows out, and place the washed skin tissue at 23 ℃ to air-dry for 31.2 hours for later use.

[0048] S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue was enzymatically hydrolyzed with pepsin solution, and the pH value was adjusted to complete the preliminary digestion; the enzymatic hydrolyzate was centrifuged to collect the supernatant, and a salt solution was added for salting out, and the precipitate was collected by centrifugation. The detailed process is as follows: the air-dried skin tissue was placed in a 0.22% mass fraction pepsin solution, the pH of the solution was adjusted to 2.04, and stirring was maintained at 38 °C. The enzymatic hydrolysis reaction time was 5.2 h. After the enzymatic hydrolysis was completed, the pH was adjusted to 8.1 with 0.52 mol / L acetic acid solution, and then adjusted to 2.04, and allowed to stand for 16 min to ensure complete digestion. The enzymatic hydrolyzed solution was centrifuged at 8200 r / min for 16 min, the supernatant was collected, and 5.2 mol / L NaCl solution was slowly added to the supernatant. The supernatant and NaCl solution were mixed at a molar ratio of 1.04:1, stirred until a flocculent precipitate was formed, and then centrifuged again at 8200 r / min for 16 min, and the resulting precipitate was collected for later use.

[0049] S3. Purification and preparation of collagen solution: The precipitate is dialyzed to remove impurities, and freeze-dried to obtain a crude collagen extract; the crude extract is dissolved in an acidic solution to prepare a collagen solution, and stirred until it is uniform and transparent. The detailed process is: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 11600Da, deionized water is used as the dialysis medium, and the dialysis is performed at 4.4 ℃ for 43.2h, and the dialysis fluid is replaced every 5.2 hours until the impurities are completely removed. After the dialysis is completed, the dialysis fluid is transferred to a freeze dryer and freeze-dried at -51 ℃ to obtain a crude collagen extract. The freeze-dried crude extract is dissolved in a 0.52mol / L acetic acid solution to prepare a collagen solution with a concentration of 1.04%, and stirred at a stirring rate of 310 r / min for 32min until the solution is uniform and transparent, and the collagen solution is obtained for use.

[0050] S4. Enzymatic hydrolysis and separation and purification: proteases are added to the collagen solution for enzymatic hydrolysis, and the enzymes are inactivated after completion; the enzymatic hydrolysis products are centrifuged and filtered, separated and purified using a molecular weight cutting membrane, and freeze-dried to obtain giant salamander peptide powder. The detailed process is as follows: alkaline protease is added to the collagen solution for enzymolysis, and the enzymolysis conditions are as follows: enzymolysis time 4.2h, temperature 50.4℃, pH value 8.04, enzyme dosage 6100U / g, and solid-liquid ratio 0.10g / mL; after the enzymolysis is completed, the reaction solution is placed in a 96℃ water bath and heated for 10.4min to inactivate the enzyme activity, and the enzymolysis product after enzyme inactivation is centrifuged at a speed of 8200r / min for 16min, and the supernatant is collected and filtered through a 0.45μm filter membrane. The filtered solution is separated and purified by cutting the membrane, and collagen peptide solutions of different molecular weight ranges are collected respectively. The collagen peptide solutions of each molecular weight range are diluted to an appropriate concentration, transferred to a freeze dryer, and freeze-dried at -51℃ to obtain giant salamander peptide powders of different molecular weights; The method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 540 parts by weight of benzene were added to a reactor, 1.6 parts of anionic initiator sec-butyl lithium were added to the reactor via a syringe in a glove box, 68 parts of isoprene were added to the reactor, and the reaction was continued for 10.4 hours, then 130 parts of styrene were added to the reactor via a syringe in the glove box, and the polymerization was continued for 38.4 hours, then benzene was removed from the reactor, and diphenylethylene in an amount 10 times the molar amount of sec-butyl lithium was added, and 560 parts of isoprene were added to the reactor. mL of tetrahydrofuran was directly distilled into the reactor, and then the reactor was cooled to -70.8°C, and then 42 parts of 4-vinylpyridine were added, and the reaction was continued for 78 minutes. Then, 8 parts of methanol were used to terminate the triblock copolymer, and the triblock copolymer was dissolved in chloroform, and then added dropwise to excess methanol for two precipitation purifications. The precipitate was collected by filtration and washed with methanol 3 times. Finally, the purified copolymer was placed in a vacuum drying oven and dried at 46°C for 38.4 hours until the solvent was completely removed, and finally isoprene-styrene-4-vinylpyridine (ISV) block copolymer was obtained.

[0051] A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1:5.2. The concentration of the glycerol solution was 10.4 wt.%. The solvent was tetrahydrofuran and 1,4-dioxane in a mass ratio of 4:7.2. All raw materials were placed in a stirring device with a stirring rate of 410 rpm for 72 min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 13.2 h to ensure that the glycerol and block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer. The liquid film thickness was adjusted to 238 μm. The coated substrate was quickly transferred to a deionized water precipitation bath. The precipitation bath temperature was maintained at 23.8 ℃, soaked for 13.8 min to remove the solvent and glycerol and form a complete mesoporous membrane structure. Subsequently, the membrane was taken out from the deionized water and rinsed with deionized water for 3 times, each rinse time was 10.4 min, to completely remove the residual solvent and glycerol, and finally obtain the mesoporous membrane.

[0052] A3: Regulation of membrane pore size: The mesoporous membrane was immersed in a citric acid-sodium citrate buffer solution with a pH of 3.38. The stirring rate of the buffer solution was 204 rpm and the immersion time was 78 min. The cut membrane was then rinsed with deionized water for 4 times and placed in a 53 °C vacuum drying oven for 18.4 h until the membrane was completely dry to obtain the cut membrane.

[0053] The giant salamander peptide is an alkaline protease collagen peptide; the average molecular weight of the giant salamander peptide is 3080 Da; the polydispersity index of the giant salamander peptide is 1.38.

[0054] Example 4

[0055] A method for preparing a giant salamander peptide comprises the following steps: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use. The detailed process is: Use healthy farmed giant salamanders as raw materials, select individuals with an average weight of 6kg and a body length of 75cm, place the giant salamanders in a 16 ℃ environment, anesthetize them with a MS-222 solution with a concentration of 0.7 g / L, dissect them, obtain skin tissue, cut the skin tissue into small pieces of about 6mm × 6mm, and then place the small pieces in a solvent mixed with ethanol and deionized water at a volume ratio of 1.5:1, soak them at 30 ℃ for 14h until fat and impurities are removed. After soaking, wash the skin tissue repeatedly with deionized water until no obvious color flows out, and place the washed skin tissue at 25 ℃ to air-dry for 36h for later use.

[0056] S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue was enzymatically hydrolyzed with pepsin solution, and the pH value was adjusted to complete the preliminary digestion; the enzymatic hydrolyzate was centrifuged to collect the supernatant, and a salt solution was added for salting out, and the precipitate was collected by centrifugation. The detailed process is as follows: the air-dried skin tissue was placed in a 0.3% mass fraction pepsin solution, the pH of the solution was adjusted to 2.2, and stirring was maintained at 40 °C. The enzymatic hydrolysis reaction time was 6 hours. After the enzymatic hydrolysis was completed, the pH was adjusted to 8.5 with 0.6 mol / L acetic acid solution, and then adjusted to 2.2, and allowed to stand for 20 minutes to ensure complete digestion. The enzymatic hydrolyzed solution was centrifuged at 9000r / min for 20 minutes, the supernatant was collected, and 6 mol / L NaCl solution was slowly added to the supernatant. The supernatant and NaCl solution were mixed at a molar ratio of 1.2:1, stirred until a flocculent precipitate was formed, and then centrifuged again at 9000r / min for 20 minutes, and the resulting precipitate was collected for later use.

[0057] S3. Purification and preparation of collagen solution: The precipitate is dialyzed to remove impurities, and lyophilized to obtain a crude collagen extract; the crude extract is dissolved in an acidic solution to prepare a collagen solution, and stirred until it is uniform and transparent. The detailed process is as follows: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 14000Da, deionized water is used as the dialysis medium, and the dialysis is performed at 6 ℃ for 48 hours, and the dialysis fluid is replaced every 6 hours until the impurities are completely removed. After the dialysis is completed, the dialysis fluid is transferred to a freeze dryer and lyophilized at -55 ℃ to obtain a crude collagen extract. The lyophilized crude extract is dissolved in a 0.6 mol / L acetic acid solution to prepare a collagen solution with a concentration of 1.2%, and stirred at a stirring rate of 350r / min for 40 minutes until the solution is uniform and transparent, and the collagen solution is obtained for standby use.

[0058] S4. Enzymatic hydrolysis and separation and purification: proteases were added to the collagen solution for enzymatic hydrolysis, and the enzymes were inactivated after completion; the enzymatic hydrolysis products were centrifuged and filtered, separated and purified using a molecular weight cutting membrane, and freeze-dried to obtain giant salamander peptide powder. The detailed process is: trypsin was added to the collagen solution for enzymatic hydrolysis, and the enzymatic hydrolysis conditions were: enzymatic hydrolysis time 5h, temperature 42 ℃, pH value 8.2, enzyme dosage 6500U / g, and solid-liquid ratio 0.12g / mL. After the enzymatic hydrolysis was completed, the reaction solution was placed in a 100 ℃ water bath and heated for 12min to inactivate the enzyme activity. The enzymatic hydrolysis product after enzyme inactivation was centrifuged at a speed of 9000 r / min for 20min, the supernatant was collected, and filtered through a 0.45μm filter membrane. The filtered solution was separated and purified by a cutting membrane, and collagen peptide solutions of different molecular weight ranges were collected respectively. The collagen peptide solutions of each molecular weight range were diluted to an appropriate concentration, transferred to a freeze dryer, and freeze-dried at -55 ℃ to obtain giant salamander peptide powders of different molecular weights; The method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 600 parts by weight of benzene were added to a reactor, 2.0 parts of anionic initiator sec-butyl lithium were added to the reactor via a syringe in a glove box, 80 parts of isoprene were added to the reactor, and the reaction was continued for 12 hours, then 150 parts of styrene were added to the reactor via a syringe in the glove box, and the polymerization was continued for 40 hours, then benzene was removed from the reactor, and diphenylethylene in an amount 10 times the molar amount of sec-butyl lithium was added, and 600 parts of isoprene were added to the reactor. mL of tetrahydrofuran was directly distilled into the reactor, and then the reactor was cooled to -78°C, and then 50 parts of 4-vinylpyridine were added, and the reaction was continued for 90 minutes. Then, 10 parts of methanol were used to terminate the triblock copolymer, and the triblock copolymer was dissolved in chloroform, and then added dropwise to excess methanol for two precipitation purifications. The precipitate was collected by filtration and washed with methanol 3 times. Finally, the purified copolymer was placed in a vacuum drying oven and dried at 50°C for 48 hours until the solvent was completely removed, and finally isoprene-styrene-4-vinylpyridine (ISV) block copolymer was obtained.

[0059] A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1:6.0, the concentration of the glycerol solution was 12.0 wt.%, and the solvent was tetrahydrofuran and 1,4-dioxane in a mass ratio of 4:8.0. All raw materials were placed in a stirring device with a stirring rate of 450 rpm and stirred for 80 min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 14 h to ensure that the glycerol and the block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer, and the liquid film thickness was adjusted to 250 μm. The coated substrate was quickly transferred to a deionized water precipitation bath, and the precipitation bath temperature was maintained at 25 °C. It was soaked for 15 min to remove the solvent and glycerol to form a complete mesoporous membrane structure. Subsequently, the membrane was taken out of the deionized water and rinsed with deionized water 4 times, each rinsing time was 12 min, to completely remove the residual solvent and glycerol, and finally a mesoporous membrane was obtained.

[0060] A3: Regulation of membrane pore size: The mesoporous membrane was immersed in a citric acid-sodium citrate buffer solution with a pH of 3.5. The stirring rate of the buffer solution was 220 rpm and the immersion time was 90 min. The cut membrane was then rinsed with deionized water for 5 times and placed in a 55 °C vacuum drying oven for 20 h until the membrane was completely dry to obtain the cut membrane.

[0061] The giant salamander peptide is a trypsin collagen peptide; the average molecular weight of the giant salamander peptide is in the range of 5000 Da; and the polydispersity index of the giant salamander peptide is 1.5.

[0062] Comparative Example 1 The method is basically the same as Example 1, except that the pH value during the enzymatic hydrolysis is 6.0, which exceeds the range of 6.8 to 7.2 for the enzymatic hydrolysis of neutral protease.

[0063] Comparative Example 2 The method is basically the same as Example 1, except that dialysis is not used to remove impurities.

[0064] Comparative Example 3 It is basically the same as Example 1, except that NaCl is not used for salting out.

[0065] Comparative Example 4 The method is basically the same as Example 1, except that glycerol is not used to induce phase separation during the preparation of the cut membrane.

[0066] Comparative Example 5 The difference is that the amount of protease enzyme used is reduced by 50%.

[0067] Comparative Example 6 The method is substantially the same as Example 1, except that the concentration of the glycerol solution is reduced to 5.0 wt.%.

[0068] Comparative Example 7 The method is substantially the same as Example 1, except that the mass ratio of tetrahydrofuran to 1,4-dioxane is adjusted to 2:8.

[0069] Comparative Example 8 The method is basically the same as Example 1, except that the citric acid-sodium citrate buffer solution is not used to adjust the membrane pore size.

[0070] Performance Test: Dispersibility index test: This experiment aims to determine the uniformity of the molecular weight distribution of giant salamander peptides, namely the polydispersity index (PDI), and evaluate its uniformity. First, 10 mg of giant salamander peptide was dissolved in 1 mL of deionized water and ultrasonically shaken for 5 minutes to ensure full dissolution. Subsequently, gel permeation chromatography (GPC-SEC) analysis was performed using polyethylene glycol (PEG) or sodium polystyrene sulfonate (PSS) as standards to calculate the number average molecular weight (Mn) and weight average molecular weight (Mw), and the polydispersity index was calculated using the formula PDI =Mw / Mn. Ideally, the PDI should be close to 1.2. If the PDI exceeds 1.5, it indicates that the molecular weight distribution is wide and uneven, which may affect the stability and biological activity of the product.

[0071] Molecular weight distribution analysis: This experiment is used to further accurately determine the molecular weight range of giant salamander peptides to ensure that they meet the target range of 200-5000 Da. First, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to determine the molecular weight distribution spectrum of giant salamander peptides, observe the distribution of major peptide segments, and calculate whether the main peak molecular weight meets the standard. In addition, the samples were separated by high-performance liquid chromatography (HPLC), using the reversed-phase HPLC (RP-HPLC) method, using a C18 chromatographic column and gradient elution mode, recording the retention time of each peak, and calculating the proportion of each molecular weight component. If the components below 200 Da or above 5000 Da exceed 10%, it means that the screening accuracy is insufficient and the purification process needs to be optimized.

[0072] Solubility test: This experiment is used to evaluate the solubility of giant salamander peptide to ensure its application potential in food, medicine and cosmetics. First, 10 mg of giant salamander peptide was dissolved in 1 mL of deionized water, the dissolution was observed, and the absorbance at 600 nm (OD600) was measured using a spectrometer. If OD600 was greater than 0.2, it indicated poor solubility. In addition, 10 mg of giant salamander peptide was dissolved in buffer solutions of pH 3, pH 7, and pH 10, respectively, to observe its solubility in different acidic and alkaline environments. Finally, the dissolution of the sample was recorded within 10 s, 30 s, 60 s, and 120 s, and the average dissolution time was calculated. If the complete dissolution time exceeded 2 minutes, it indicated poor solubility.

[0073] Biological activity test: This experiment mainly tests the antioxidant and cell proliferation promoting effects of giant salamander peptides. The antioxidant test uses the DPPH free radical scavenging experiment to calculate the IC50 (half inhibitory concentration). If the IC50 is lower than 0.2 mg / mL, it means that the antioxidant capacity is strong. In addition, the MTT or CCK-8 cell experiment is used to detect the proliferation effect of giant salamander peptides on fibroblasts (such as NIH-3T3 cells). Under the concentration conditions of 0, 10, 50, 100 and 200 μg / mL, the cell proliferation rate is calculated. If the cell proliferation rate of the 100 μg / mL treatment group exceeds 120%, it indicates that the giant salamander peptide has a good cell growth promoting effect and is suitable for the fields of medicine and cosmetics.

[0074] Thermal stability: This experiment aims to test the stability of giant salamander peptide at different temperatures to evaluate its suitability in food, medicine and cosmetics. First, take an equal amount of giant salamander peptide solution (1 mg / mL) and place it in a water bath at 25 ℃, 50 ℃, 75 ℃ and 100 ℃ for 30 minutes. Subsequently, circular dichroism (CD) was used to observe the changes in its secondary structure, and high performance liquid chromatography (HPLC) was used to detect the changes in molecular weight and calculate the degradation rate. If the giant salamander peptide still maintains more than 90% integrity after treatment at 100 ℃, it means that it has good heat resistance and is suitable for various high temperature processing environments.

[0075] The properties of the giant salamander peptides of Examples 1 to 4 and Comparative Examples 1 to 8 are summarized in Table 1.

[0076] Table 1 Performance of the giant salamander peptides of Examples 1 to 4 and Comparative Examples 1 to 8

[0077] Under the optimal process conditions, the dispersity index (PDI) of giant salamander peptides was maintained at 1.2~1.5, ensuring a high molecular weight uniformity. When the enzymatic pH was out of control, glycerol was not used to induce phase separation, the glycerol concentration was reduced, or the solvent ratio was adjusted, the PDI increased, indicating that the molecular weight distribution became wider and the uniformity decreased. Dialysis treatment is essential for improving solubility. If dialysis treatment is not performed, the OD600 of the solution will increase significantly, indicating that the impurity residue affects the solubility, and improper membrane pore size regulation will also lead to a decrease in solubility. In addition, the antioxidant capacity is greatly affected by impurities and membrane pore size. Failure to dialyze or improper membrane pore size regulation will lead to an increase in IC50 and a decrease in the antioxidant effect. The reduction in the amount of protease may help retain some functional peptides, thereby increasing the cell proliferation rate. In terms of thermal stability, insufficient dialysis or uneven membrane pore size will reduce the heat resistance of giant salamander peptides and reduce their structural retention rate under high temperature conditions.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that all equivalent structural changes made under the concept of the present invention and using the contents of the present invention specification and drawings should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a giant salamander peptide, characterized in that: The following steps are involved: S1. Raw material processing: Select healthy artificially cultivated giant salamanders, dissect and obtain skin tissue, and cut it into small pieces; soak the skin tissue in a mixed solvent of ethanol and deionized water for degreasing and decolorization, wash and air-dry for later use; S2. Preliminary enzymatic hydrolysis and separation: The air-dried skin tissue is enzymatically hydrolyzed with a pepsin solution and the pH value is adjusted to complete the preliminary digestion; The enzymatic hydrolysate is centrifuged to collect the supernatant, a salt solution is added for salting out, and the precipitate is collected by centrifugation; S3. Purification and preparation of collagen solution: dialyze the precipitate to remove impurities, and freeze-dry to obtain a crude collagen extract; Dissolve the crude extract in an acidic solution to prepare a collagen solution, and stir until it becomes uniform and transparent; S4. Enzymatic hydrolysis and separation and purification: proteases are added to the collagen solution for enzymatic hydrolysis, and the enzymes are inactivated after completion; the enzymatic hydrolysis products are centrifuged and filtered, separated and purified using a molecular weight cutting membrane, and freeze-dried to obtain giant salamander peptide powder.

2. The method for preparing a giant salamander peptide according to claim 1, characterized in that: The detailed process of the S1 raw material processing is as follows: using healthy farmed giant salamanders as raw materials, selecting individuals with an average body weight of 4-6 kg and a body length of 70-75 cm, placing the giant salamanders in an environment of 12-16°C, anesthetizing them with a MS-222 solution with a concentration of 0.5-0.7 g / L, dissecting them, obtaining skin tissue, cutting the skin tissue into small pieces of about 4 mm × 4 mm~6 mm × 6 mm, then placing the obtained small pieces in a solvent mixed with ethanol and deionized water in a volume ratio of (1.0-1.5):1, soaking them at 20-30°C for 10-14 hours until fat and impurities are removed, after soaking, repeatedly washing the skin tissue with deionized water until no obvious color flows out, and air-drying the washed skin tissue at 20-25°C for 24-36 hours for later use.

3. The method for preparing a giant salamander peptide according to claim 1, characterized in that: The detailed process of the preliminary enzymatic hydrolysis and separation of S2 is as follows: placing the air-dried skin tissue in a 0.1~0.3% mass fraction pepsin solution, adjusting the solution pH to 1.8~2.2, and stirring at 35~40°C. The enzymatic hydrolysis reaction time is 4~6h. After the enzymatic hydrolysis is completed, the pH is adjusted to 7.5~8.5 with 0.4~0.6mol / L acetic acid solution, and then adjusted to 1.8~2.2, and allowed to stand for 10~20min to ensure complete digestion. The enzymatic hydrolysis solution is centrifuged at a speed of 7000~9000 r / min for 10~20min, the supernatant is collected, and 4~6 mol / L NaCl solution is slowly added to the supernatant. The supernatant and NaCl solution are mixed at a molar ratio of (0.8~1.2):1, stirred to form a flocculent precipitate, and then centrifuged again at a speed of 7000~9000 r / min for 10~20min, and the resulting precipitate is collected for later use.

4. The method for preparing a giant salamander peptide according to claim 1, characterized in that: The detailed process of S3 purification and collagen solution preparation is as follows: the collected precipitate is placed in a dialysis bag with a molecular weight cutoff range of 8,000 to 14,000 Da, deionized water is used as the dialysis medium, and the dialysis is performed at 2 to 6 ° C for 36 to 48 hours, and the dialysis fluid is replaced every 4 to 6 hours until the impurities are completely removed. After the dialysis is completed, the dialysis fluid is transferred to a freeze dryer and freeze-dried at -45 to -55 ° C to obtain a crude collagen extract. The freeze-dried crude extract is dissolved in a 0.4 to 0.6 mol / L acetic acid solution to prepare a collagen solution with a concentration of 0.8 to 1.2%, and stirred at a stirring rate of 250 to 350 r / min for 20 to 40 minutes until the solution is uniform and transparent, and the collagen solution is obtained for use.

5. The method for preparing a giant salamander peptide according to claim 1, characterized in that: The detailed process of the S4 enzymatic hydrolysis and separation and purification is as follows: adding protease to the collagen solution for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, the reaction solution is placed in a 90-100°C water bath and heated for 8-12 minutes to inactivate the enzyme activity; the enzymatic hydrolysis product after enzyme inactivation is centrifuged at a speed of 7000-9000 r / min for 10-20 minutes; the supernatant is collected and filtered through a 0.45 μm filter membrane; the filtered solution is separated and purified by cutting the membrane; collagen peptide solutions of different molecular weight ranges are collected respectively; each collagen peptide solution of each molecular weight range is diluted to an appropriate concentration, transferred to a freeze dryer, and freeze-dried at -45--55°C to obtain giant salamander peptide powders of different molecular weights.

6. The method for preparing a giant salamander peptide according to claim 5, characterized in that: The protease is any one of neutral protease, pepsin, alkaline protease and trypsin; When the protease is a neutral protease, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 48~52℃, pH value 6.8~7.2, enzyme dosage 5500~6500 U / g, solid-liquid ratio 0.08~0.12 g / mL; When the protease is pepsin, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 35~39℃, pH value 1.8~2.2, enzyme dosage 5500~6500 U / g, solid-liquid ratio 0.08~0.12 g / mL; When the protease is alkaline protease, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3-5 hours, temperature 48-52 °C, pH value 7.8-8.2, enzyme dosage 5500-6500 U / g, solid-liquid ratio 0.08-0.12 g / mL; When the protease is trypsin, the enzymatic hydrolysis conditions are: enzymatic hydrolysis time 3~5h, temperature 38~42℃, pH value 7.8~8.2, enzyme dosage 5500~6500 U / g, and solid-liquid ratio 0.08~0.12 g / mL.

7. The method for preparing a giant salamander peptide according to claim 5, characterized in that: The method for preparing the cutting film comprises the following steps: A1: Preparation of isoprene-styrene-4-vinylpyridine block copolymer: 450-600 parts by weight of benzene are added to a reactor, 1.0-2.0 parts of anionic initiator sec-butyl lithium are added to the reactor via a syringe in a glove box, 50-80 parts of isoprene are added to the reactor, and the reaction is continued for 8-12 hours, and then 100-150 parts of styrene are added to the reactor via a syringe in the glove box, and the polymerization is carried out for 36-40 hours, and then benzene is removed from the reactor, and diphenylethylene is added in an amount 10 times the molar amount of sec-butyl lithium, 500-600 mL of tetrahydrofuran is directly distilled into the reactor, and then the reactor is cooled to -60--78°C, and then 30-50 parts of 4-vinylpyridine are added, and the reaction is continued for 60-90 minutes, followed by 5-10 The triblock copolymer was terminated by adding 1 part of methanol, the triblock copolymer was dissolved in chloroform, and then dropped into excess methanol for two precipitation purifications, the precipitate was collected by filtration, and washed with methanol three times, and finally, the purified copolymer was placed in a vacuum drying oven and dried at 40-50°C for 24-48 h until the solvent was completely removed, and finally an isoprene-styrene-4-vinylpyridine (ISV) block copolymer was obtained; A2: Preparation of mesoporous membrane: The prepared isoprene-styrene-4-vinylpyridine (ISV) block copolymer was mixed with glycerol solution in a mass ratio of 1: (4.0~6.0), the concentration of the glycerol solution was 8.0~12.0 wt.%, and the solvent was tetrahydrofuran and 1,4-dioxane in a mass ratio of 4: (6.0~8.0). All raw materials were placed in a stirring device with a stirring rate of 350~450 rpm for 60~80 min. The resulting solution was transferred to a sealed container and continued to be stirred at room temperature for 12~14 h to ensure that the glycerol and block copolymer were fully mixed to form a stable casting solution. The prepared casting solution was evenly coated on the surface of the glass substrate support layer, and the liquid film thickness was adjusted to 220~250 μm. The coated substrate was quickly transferred to a deionized water precipitation bath, and the precipitation bath temperature was maintained at 22~25 ℃, soak for 12-15 minutes to remove the solvent and glycerol, forming a complete mesoporous membrane structure, then take the membrane out of the deionized water and rinse it with deionized water for 3-4 times, each rinsing time is 8-12 minutes, to completely remove the residual solvent and glycerol, and finally obtain a mesoporous membrane; A3: Regulation of membrane pore size: The mesoporous membrane was immersed in a citric acid-sodium citrate buffer solution with a pH of 3.2-3.

5. The stirring rate of the buffer solution was 180-220 rpm and the immersion time was 60-90 min. The cut membrane was then rinsed with deionized water for 4-5 times and placed in a vacuum drying oven at 50-55 °C for 16-20 h until the membrane was completely dry to obtain the cut membrane.

8. A giant salamander peptide, characterized in that: The giant salamander peptide is prepared by the preparation method according to any one of claims 1 to 7; The giant salamander peptide is any one of neutral protease collagen peptide, pepsin collagen peptide, alkaline protease collagen peptide or trypsin collagen peptide; The average molecular weight of the giant salamander peptide is in the range of 200 to 5000 Da; The polydispersity index of the giant salamander peptide is 1.2-1.

5.

9. Use of the giant salamander peptide as claimed in claim 8 in the fields of functional health products, medicine and functional cosmetics.

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