Collagen freeze-dried powder as well as preparation method and application thereof

Abstract

The invention provides collagen freeze-dried powder as well as a preparation method and application thereof, and belongs to the technical field of protein industry. The method comprises the following three steps: crude extraction, fine extraction and freeze-drying. In the crude extraction step, collagen suspension is obtained by mixing, heating, emulsifying and the like. In the step of fine extraction, the collagen is extracted and purified by means of homogenization, microwave treatment and the like. In the freeze-drying step, polyether derivatives and dopamine analogues are added, and collagen freeze-dried powder is obtained through the processes of reaction, centrifugation, drying and the like. According to the method, the yield and purity of collagen can be greatly improved, and meanwhile, the method is relatively good in environmental protection and economical efficiency. The method can be widely applied to preparation of antioxidants, cosmetics, food additives and the like, and has important significance and good application prospects in the field of protein industry.

Description

Technical Field

[0001] The present invention relates to the technical field of protein industry, and in particular to a collagen freeze-dried powder and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant structural protein in animals and is widely found in connective tissues such as skin, bones, cartilage, and blood vessels. It consists of three polypeptide chains forming a triple helix structure, has good biocompatibility and mechanical strength, and plays an important role in maintaining tissue structural integrity and supporting cell growth.

[0003] At present, industrially produced collagen mainly comes from mammalian tissues such as cattle and pigs. The traditional preparation process usually includes alkaline treatment to remove non-collagenous proteins, extraction of soluble collagen under acidic conditions, salting out and dialysis purification. However, animal-derived collagen has the risk of pathogen contamination, unstable quality, and complex extraction process. In addition, certain groups of people have a low acceptance of animal-derived products.

[0004] To overcome the above limitations, researchers have tried to use genetic engineering technology to develop recombinant human collagen. By expressing collagen genes in host cells such as Escherichia coli, yeast, insects, mammals, and plants, recombinant proteins similar in structure and function to natural collagen can be obtained. Different expression systems have their own advantages and disadvantages. For example, Escherichia coli has high expression levels but lacks post-translational modification, and mammalian cells are fully modified but have low yields and high costs. Currently, recombinant collagen used commercially mainly comes from yeast and insect cell systems.

[0005] In addition to direct expression, soluble recombinant collagen peptides can also be constructed through genetic engineering. These peptides retain the key structural domains of natural collagen and have biological functions such as fiber formation, gel formation, and promotion of cell adhesion and growth. They have broad application prospects in the fields of tissue engineering scaffold materials, wound dressings, and cosmetic additives. However, their mechanical strength and thermal stability are not as good as those of complete collagen molecules.

[0006] Hydrolysis is another important way to prepare collagen polypeptides. Animal-derived collagen can be degraded into polypeptide fragments with smaller molecular weight by enzymatic hydrolysis, acid-base hydrolysis and other methods, which are called hydrolyzed collagen or gelatin. Hydrolyzed collagen has good water solubility and bioavailability, and is beneficial to skin, bone and joint health as a nutritional supplement. However, its structure and function are quite different from those of natural collagen.

[0007] In recent years, with the development of synthetic biology and protein engineering technology, directed evolution and rational design strategies have been used to optimize the yield and performance of recombinant collagen. The yield and post-translational modification level of recombinant collagen can be improved by screening efficient expression hosts, optimizing codons, introducing auxiliary factors, etc. In addition, site-directed mutagenesis and chimera technology can give recombinant collagen new biological functions, such as regulating degradation rate and introducing cell adhesion sites. This provides ideas for the development of new controlled release carriers, bionic scaffold materials, etc.

[0008] In general, after more than 30 years of development, recombinant collagen technology has become mature and provides a substitute for animal-derived collagen. However, due to factors such as expression system, output, and cost, its application in the fields of biomedicine and functional foods still faces challenges. In the future, with the advancement of emerging technologies such as synthetic biology, it is expected to break through bottlenecks and achieve large-scale production and wide application of recombinant collagen. At the same time, the quality safety, immunogenicity, and clinical efficacy of recombinant collagen still need to be deeply evaluated. Summary of the invention

[0009] In view of this, the present invention provides a collagen freeze-dried powder and a preparation method and application thereof, which comprises the following steps by weight:

[0010] (1) Crude extraction: First, 0.1-0.5 parts of an emulsifier and 30-50 parts of fresh fish skin are added to a feed tank; then, 100-160 parts of deionized water are added to the feed tank, mixed and stirred, and heated in a water bath to 60-80° C. and kept constant for 2-6 hours; then, 15-20 parts of an acrylate derivative and 1-3 parts of a polymerization inhibitor are added to the feed tank, and mechanical stirring and emulsification are performed for 15-30 minutes; finally, 10-20 parts of a stabilizer are added, reacted at 35-50° C. for 8-12 hours, and ultrasonicated for 2-4 hours to obtain a suspension, and then 10-20 parts of the suspension are dispersed in 100-120 parts of anhydrous ethanol, and then the ethanol is evaporated at 50-70° C. for 4-6 hours to obtain a crude extraction product;

[0011] (2) Fine extraction: 4-7 parts of weak base, 1-2 parts of dry chloride and 1-2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 20-40 min under a nitrogen atmosphere, wherein the microwave power is 200-400 W, and the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a fine extraction product;

[0012] (3) Freeze-dried product: Add 2-5 parts of the crude extracted product obtained in step (2) to 20-40 parts of the polyether derivative, then ultrasonically treat to form a suspension, then add 1-3 parts of dopamine analogs, stir evenly, add ammonium salt solution dropwise at a uniform rate, stir and react at 40-60° C. for 2-3 hours, then centrifuge at 120,000-160,000 rpm for 20-30 minutes to collect the organic phase, add twice the volume of 4° C. acetone, ultrasonically mix, and place in an oven for drying to obtain a dry powder.

[0013] The preparation method of the collagen freeze-dried powder comprises the following steps: in step (1), the emulsifier is one of glyceryl monostearate, sucrose fatty acid ester, soybean lecithin, sorbitan monostearate, and polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is one of aminoacrylate, 3-aminoacrylate methyl ester, and aminoacrylate dodecyl ester amide; the inhibitor is one of nano titanium dioxide, nano magnesium oxide, and nano sodium chloride; and the stabilizer is one of potassium sulfate, dibutyltin dilaurate, and dioctyltin thioglycolate.

[0014] In the preparation method of the collagen freeze-dried powder, the rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 250-350 W, and the temperature of the ultrasonication is below 15°C.

[0015] In the preparation method of the collagen freeze-dried powder, the weak base in step (2) is one of sodium bicarbonate and ammonium hydroxide; the chloride is one of magnesium chloride, zinc chloride, and calcium chloride; the buffer is one of PBS, Tris-HCl, and sodium acetate; and the acid rinse is hydrochloric acid rinse, wherein the concentration of the hydrochloric acid is 1 mM.

[0016] In the preparation method of the collagen freeze-dried powder, the pressure of the homogenization treatment in step (2) is 1MPa-3MPa, the time of the homogenization treatment is 2h-4h; the flow rate of the nitrogen is 100mL / min-200mL / min; and the temperature of the drying is 40-60°C.

[0017] In the preparation method of the collagen freeze-dried powder, the polyether derivative in step (3) is one of polyetheretherketone, polyetherimide, polyethersulfone, and polyethersulfoneetherketone; the dopamine analog is one of dopamine hydrochloride, dopamine-3-phosphate, and mercaptopolyethylene glycol dopamine; the ammonium salt solution is one of ammonium chloride solution, ammonium carbonate solution, and ammonium phosphate, and the concentration of the ammonium salt solution is 10mM.

[0018] The preparation method of the collagen freeze-dried powder is as follows: the ultrasonic method in step (3) is water bath ultrasonication, the ultrasonic power is 80-250W; the stirring speed is 180rpm; the uniform dripping rate is 0.1-0.3 parts / min, and the total amount of the uniformly dripped solution is 4-8 parts; the ultrasonic mixing temperature is 40-50°C, the ultrasonic mixing power is 100-300W; the oven drying temperature is 45-60°C.

[0019] The dry powder is prepared by adopting the preparation method of the collagen freeze-dried powder described above.

[0020] The above-mentioned collagen freeze-dried powder is used in the preparation of antioxidant preparations.

[0021] Based on the information provided, the main technical effects of this invention can be summarized as follows:

[0022] 1. High yield and high purity: The yield of the lyophilized collagen powder prepared in the examples is between 93.4% and 94.2%, and the purity is between 98.2% and 99.2%, which are much higher than the results of the comparative examples.

[0023] 2. Excellent ACE inhibitory activity: The ACE inhibitory activity (IC50 value) of the example product is between 0.042-0.059 mg / mL, which is significantly better than the comparative example product. This indicates that the product has good blood pressure lowering potential.

[0024] 3. Optimization of key process parameters:

[0025] - Addition of acrylate derivatives significantly improves product quality and activity.

[0026] -Using a weak base rather than a strong base helps maintain product activity.

[0027] -Polyether derivatives and dopamine analogs are used to improve product performance.

[0028] -Using acetone instead of pure water in the final processing steps improves product quality.

[0029] 4. Potential application value: Due to its good ACE inhibitory activity, this product may be used to develop dietary therapy for patients with hypertension.

[0030] 5. Controllability and repeatability of the process: By specifying the parameters of each step (such as temperature, time, pH value, etc.) in detail, the controllability of the process and the consistency of product quality are guaranteed.

[0031] In general, this technology successfully prepared high-quality, highly active collagen freeze-dried powder by optimizing the extraction and purification process, which has potential health and medical application value.

[0032] Advantages of the present invention will be partially set forth in the following description, and partly will be obvious from the description, or may be learned through implementation of the embodiments of the present invention. DETAILED DESCRIPTION

[0033] The following is a preferred embodiment of the present invention. It should be noted that a person skilled in the art may make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

[0034] It should be noted that the species of the fresh fish skin involved in the present invention is silver carp. In addition, the weight portion involved in the present invention is measured in kg (kilogram).

[0035] Example 1

[0036] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0037] (1) Crude extraction: First, add 0.1 parts of emulsifier and 50 parts of fresh fish skin to the feed tank; then, add 100 parts of deionized water to the feed tank, mix and stir, and heat to 80°C in a water bath and keep constant for 2 hours; then, add 15 parts of acrylate derivatives and 3 parts of inhibitors to the feed tank, mechanically stir and emulsify for 15 minutes; finally, add 20 parts of stabilizer, react at 35°C for 8 hours, ultrasonicate for 4 hours to obtain a suspension, then disperse 10 parts of the suspension in 120 parts of anhydrous ethanol, then evaporate the ethanol at 50°C for 6 hours to obtain a crude extraction product. The emulsifier in step (1) is glyceryl monostearate; the acrylate derivative is aminoacrylate; the inhibitor is nano titanium dioxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 250, and the temperature of the ultrasonication is below 15°C.

[0038] (2) Refined extraction: 4 parts of weak base, 2 parts of dry chloride and 1 part of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 40 minutes under a nitrogen atmosphere, wherein the microwave power is 200 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. The weak base in step (2) is sodium bicarbonate; the chloride is magnesium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 1 MPa, and the time of the homogenization treatment is 4 hours; the flow rate of the nitrogen is 100 mL / min; and the drying temperature is 60°C.

[0039] (3) Freeze-dried finished product: Add 5 parts of the crude extracted product obtained in step (2) to 20 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 1 part of the dopamine analog, stir evenly, uniformly add ammonium salt solution, stir and react at 60°C for 2h, then centrifuge at 160000rpm for 20min to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyetheretherketone; the dopamine analog is dopamine hydrochloride; the ammonium salt solution is ammonium chloride solution, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 80W; the stirring speed is 180rpm; the uniform dripping rate is 0.3 parts / min, and the total amount of the uniformly dripped solution is 4 parts; the ultrasonic mixing temperature is 50°C, and the ultrasonic mixing power is 100W; the oven drying temperature is 60°C.

[0040] Example 2

[0041] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0042] (1) Crude extraction: First, add 0.5 parts of emulsifier and 30 parts of fresh fish skin to the feed tank; then, add 160 parts of deionized water to the feed tank, mix and stir, and heat to 60°C in a water bath and keep constant for 6 hours; then, add 15 parts of acrylate derivatives and 3 parts of inhibitors to the feed tank, mechanically stir and emulsify for 15 minutes; finally, add 20 parts of stabilizer, react at 35°C for 12 hours, ultrasonicate for 2 hours, and prepare a suspension, then disperse 20 parts of the suspension in 100 parts of anhydrous ethanol, and then evaporate the ethanol at 70°C for 4 hours to obtain a crude extraction product. The emulsifier in step (1) is sucrose fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is dibutyltin dilaurate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 350 W, and the temperature of the ultrasonication is below 15°C.

[0043] (2) Refined extraction: Provide 7 parts of weak base, 1 part of dry chloride and 2 parts of buffer and mix with the crude extraction product obtained in step (1), and homogenize; then transfer to a sealed container, microwave for 20 minutes under a nitrogen atmosphere, the microwave power is 400W, collect the solid product and rinse with acid, then rinse with deionized water and dry to obtain a refined extraction product. The weak base in step (2) is ammonium hydroxide; the chloride is zinc chloride; the buffer is Tris-HCl; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1mM. The pressure of the homogenization treatment in step (2) is 3MPa, and the time of the homogenization treatment is 2h; the flow rate of the nitrogen is 200mL / min; the temperature of the drying is 40.

[0044] (3) Freeze-dried finished product: Add 2 parts of the crude extracted product obtained in step (2) to 40 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 3 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 40°C for 3 hours, then centrifuge at 120,000 rpm for 30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyetherimide; the dopamine analog is dopamine-3-phosphate; the ammonium salt solution is ammonium carbonate solution, and the concentration of the ammonium salt solution is 10 mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 250W; the stirring speed is 180rpm; the uniform dripping rate is 0.1 parts / min, and the total amount of the uniformly dripped solution is 8 parts; the ultrasonic mixing temperature is 40°C, and the ultrasonic mixing power is 300W; the oven drying temperature is 45°C.

[0045] Example 3

[0046] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0047] (1) Crude extraction: First, add 0.1 parts of emulsifier and 30 parts of fresh fish skin to the feed tank; then, add 100 parts of deionized water to the feed tank, mix and stir, and heat to 60°C in a water bath and keep constant for 2 hours; then, add 15 parts of acrylate derivatives and 1 part of inhibitor to the feed tank, mechanically stir and emulsify for 15 minutes; finally, add 10 parts of stabilizer, react at 35°C for 8 hours, ultrasonicate for 2 hours to obtain a suspension, then disperse 10 parts of the suspension in 100 parts of anhydrous ethanol, then evaporate the ethanol at 50°C for 4 hours to obtain a crude extraction product. The emulsifier in step (1) is soybean lecithin; the acrylate derivative is aminoacrylic acid dodecyl ester amide; the inhibitor is nano sodium chloride; and the stabilizer is dioctyltin thioglycolate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 250 W, and the temperature of the ultrasonication is below 15°C.

[0048] (2) Refined extraction: Provide 4 parts of weak base, 1 part of dry chloride and 1 part of buffer and mix with the crude extraction product obtained in step (1), and homogenize; then transfer to a sealed container, microwave for 20 minutes under a nitrogen atmosphere, the microwave power is 200W, collect the solid product and rinse with acid, then rinse with deionized water and dry to obtain a refined extraction product. The weak base in step (2) is sodium bicarbonate; the chloride is magnesium chloride; the buffer is one of sodium acetate; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1mM. The pressure of the homogenization treatment in step (2) is 1MPa, and the time of the homogenization treatment is 2h; the flow rate of the nitrogen is 100mL / min; the drying temperature is 40°C.

[0049] (3) Freeze-dried finished product: Add 2 parts of the crude extracted product obtained in step (2) to 20 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 1 part of the dopamine analog, stir evenly, uniformly add ammonium salt solution, stir and react at 40°C for 2h, then centrifuge at 120000rpm for 20min to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfone; the dopamine analog is thiol polyethylene glycol dopamine; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 80W; the stirring speed is 180rpm; the uniform dripping rate is 0.1 parts / min, and the total amount of the uniformly dripped solution is 4 parts; the ultrasonic mixing temperature is 40°C, and the ultrasonic mixing power is 100W; the oven drying temperature is 45°C.

[0050] Example 4

[0051] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0052] (1) Crude extraction: First, add 0.5 parts of emulsifier and 50 parts of fresh fish skin to the feed tank; then, add 160 parts of deionized water to the feed tank, mix and stir, and heat to 80°C in a water bath and keep constant for 6 hours; then, add 20 parts of acrylate derivative and 3 parts of inhibitor to the feed tank, mechanically stir and emulsify for 30 minutes; finally, add 20 parts of stabilizer, react at 50°C for 12 hours, ultrasonicate for 4 hours to obtain a suspension, then disperse 20 parts of the suspension in 120 parts of anhydrous ethanol, then evaporate the ethanol at 70°C for 6 hours to obtain a crude extraction product. The emulsifier in step (1) is sorbitan monostearate; the acrylate derivative is aminoacrylic acid dodecyl ester amide; the inhibitor is nano sodium chloride; and the stabilizer is dioctyltin thioglycolate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 350 W, and the temperature of the ultrasonication is below 15°C.

[0053] (2) Refined extraction: 7 parts of weak base, 2 parts of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 40 minutes under a nitrogen atmosphere, wherein the microwave power is 400 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. The weak base in step (2) is ammonium hydroxide; the chloride is calcium chloride; the buffer is sodium acetate; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 3 MPa, and the time of the homogenization treatment is 4 hours; the flow rate of the nitrogen is 200 mL / min; and the drying temperature is 60°C.

[0054] (3) Freeze-dried finished product: add 5 parts of the crude extracted product obtained in step (2) to 40 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 3 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 60°C for 3 hours, then centrifuge at 160000rpm for 30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfoneetherketone; the dopamine analog is mercaptopolyethylene glycol dopamine; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 250W; the stirring speed is 180rpm; the uniform dripping rate is 0.3 parts / min, and the total amount of the uniformly dripped solution is 8 parts; the ultrasonic mixing temperature is 50°C, and the ultrasonic mixing power is 300W; the oven drying temperature is 60°C.

[0055] Example 5

[0056] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0057] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 17 parts of acrylate derivatives and 2 parts of inhibitors to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, and then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0058] (2) Refined extraction: 6 parts of weak base, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the weak base is sodium bicarbonate; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0059] (3) Freeze-dried finished product: add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 2 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfone; the dopamine analog is dopamine hydrochloride; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0060] Comparative Example 1

[0061] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0062] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 2 parts of inhibitor to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The speed of the mixing and stirring in step (1) is 300 rpm; the speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0063] (2) Refined extraction: 6 parts of weak base, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the weak base is sodium bicarbonate; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0064] (3) Freeze-dried finished product: add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 2 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfone; the dopamine analog is dopamine hydrochloride; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0065] Comparative Example 2

[0066] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0067] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 17 parts of acrylate derivatives and 2 parts of inhibitors to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, and then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0068] (2) Refined extraction: 6 parts of alkali, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the alkali is sodium hydroxide; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0069] (3) Freeze-dried finished product: add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 2 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfone; the dopamine analog is dopamine hydrochloride; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0070] Comparative Example 3

[0071] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0072] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 17 parts of acrylate derivatives and 2 parts of inhibitors to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, and then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0073] (2) Refined extraction: 6 parts of weak base, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the weak base is sodium bicarbonate; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0074] (3) Freeze-dried finished product: add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of ether, then ultrasonically treat it to form a suspension, then add 2 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30 minutes to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. The dopamine analog in step (3) is dopamine hydrochloride; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0075] Comparative Example 4

[0076] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0077] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 17 parts of acrylate derivatives and 2 parts of inhibitors to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, and then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0078] (2) Refined extraction: 6 parts of weak base, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the weak base is sodium bicarbonate; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0079] (3) Freeze-dried finished product: Add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of the polyether derivative, then ultrasonically treat it to form a suspension, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30min to collect the organic phase, add two volumes of 4°C acetone, ultrasonically mix, and place in an oven for drying to obtain dry powder. The ammonium salt solution in step (3) is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0080] Comparative Example 5

[0081] The preparation method of collagen freeze-dried powder comprises the following steps by weight:

[0082] (1) Crude extraction: First, add 0.3 parts of emulsifier and 40 parts of fresh fish skin to the feed tank; then, add 130 parts of deionized water to the feed tank, mix and stir, and heat to 70°C in a water bath and keep constant for 4 hours; then, add 17 parts of acrylate derivatives and 2 parts of inhibitors to the feed tank, mechanically stir and emulsify for 22 minutes; finally, add 15 parts of stabilizer, react at 44°C for 10 hours, ultrasonicate for 3 hours to obtain a suspension, and then disperse 15 parts of the suspension in 110 parts of anhydrous ethanol, then evaporate the ethanol at 60°C for 5 hours to obtain a crude extraction product. The emulsifier in step (1) is polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is 3-amino methyl acrylate; the inhibitor is nano magnesium oxide; and the stabilizer is potassium sulfate. The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 300 W, and the temperature of the ultrasonication is below 15°C.

[0083] (2) Refined extraction: 6 parts of weak base, 1 part of dry chloride and 2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 30 minutes under a nitrogen atmosphere, wherein the microwave power is 300 W, the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a refined extraction product. In step (2), the weak base is sodium bicarbonate; the chloride is calcium chloride; the buffer is PBS; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM. The pressure of the homogenization treatment in step (2) is 2 MPa, and the time of the homogenization treatment is 3 hours; the flow rate of the nitrogen is 150 mL / min; and the drying temperature is 50°C.

[0084] (3) Freeze-dried finished product: add 2-5 parts of the crude extracted product obtained in step (2) to 30 parts of the polyether derivative, then ultrasonically treat it to form a suspension, then add 2 parts of dopamine analogs, stir evenly, uniformly add ammonium salt solution, stir and react at 50°C for 3 hours, then centrifuge at 140000rpm for 20-30 minutes to collect the organic phase, add two times the volume of pure water, ultrasonically mix, and place in an oven for drying to obtain dry powder. In step (3), the polyether derivative is polyethersulfone; the dopamine analog is dopamine hydrochloride; the ammonium salt solution is ammonium phosphate, and the concentration of the ammonium salt solution is 10mM. The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 150W; the stirring speed is 180rpm; the uniform dripping rate is 0.2 parts / min, and the total amount of the uniformly dripped solution is 6 parts; the ultrasonic mixing temperature is 45°C, and the ultrasonic mixing power is 200W; the oven drying temperature is 55°C.

[0085] Test plan

[0086] The yield and purity of the dry powder prepared by the above examples and comparative examples were tested according to standard YY / T1805.

[0087] Table 1

[0088]

[0089]

[0090] Method for determining ACE inhibitory activity

[0091] Take 120μL HHL substrate solution, add 20μL sample and mix well, and keep warm in a 37℃ constant temperature water bath for 10min. Then add 10μL ACE enzyme solution and react in a 37℃ constant temperature water bath for 30min, then add 150μL 1mol / L HCl to terminate the reaction to obtain a reaction solution. The reaction solution was analyzed by HPLC, and a blank control group was set up at the same time. The ACE inhibitory activity calculation formula is as follows:

[0092] ACE inhibitory activity % = (MN) / M×100%

[0093] Wherein, M is the peak area of ​​hippuric acid in the control group, and N is the peak area of ​​hippuric acid in the added sample group.

[0094] (4) Determination of half inhibitory concentration

[0095] The inhibitory activity was determined according to the ACE inhibition in vitro detection method, and a smooth curve was plotted with the concentration as the horizontal axis and the ACE inhibition rate as the vertical axis, and the IC50 value was calculated from the curve.

[0096] The above-mentioned related tests are shown in Table 2, which shows that the product prepared by the present application has a good blood pressure lowering effect and can be used as a potential dietary therapy for patients with hypertension. The information about ACE is as follows: angiotensin converting enzyme (ACE) is also called kininase II or peptidyl-carboxypeptidase. It is an enzyme bound to the membrane of vascular endothelial cells. It is formed by cutting the amino acid into two segments at the C-terminus of the peptide, and can hydrolyze the dipeptide residue at the C-terminus of the peptide chain. ACE is widely distributed in various tissues of the human body, with rich content in the epididymis, testis and lungs, among which the ACE activity of pulmonary capillary endothelial cells is the highest. It is attached to the surface of endothelial cells and can be decomposed and released into the blood circulation. ACE has two main functions: 1. Catalyze the conversion of angiotensin I into angiotensin II; 2. Inactivate bradykinin. Angiotensin converting enzyme has become an ideal target for the treatment of diseases such as hypertension, heart failure, type 2 diabetes and diabetic nephropathy due to these two functions. The main methods for determining ACE include colorimetry, enzyme coupling method, etc.

[0097] Table 2

[0098]

[0099]

[0100] Combining the data in Table 1 and Table 2, it can be analyzed that the yield and purity are high: the yield of the lyophilized collagen powder prepared in the embodiment is between 93.4% and 94.2%, and the purity is between 98.2% and 99.2%, which are much higher than the results of the comparative example.

[0101] Excellent ACE inhibitory activity: The ACE inhibitory activity (IC50 value) of the example product is between 0.042-0.059 mg / mL, which is significantly better than that of the comparative example product. This indicates that the product has good blood pressure lowering potential.

[0102] Optimization of key process parameters:

[0103] The addition of acrylate derivatives significantly improves product quality and activity.

[0104] Using a weak base rather than a strong base helps maintain product activity.

[0105] The use of polyether derivatives and dopamine analogs improves product performance.

[0106] Using acetone instead of pure water improves product quality in the final processing steps.

[0107] Potential application value: Due to its good ACE inhibitory activity, this product is used to develop dietary therapy for patients with hypertension.

[0108] Process controllability and repeatability: By specifying the parameters of each step (such as temperature, time, pH value, etc.) in detail, the controllability of the process and the consistency of product quality are guaranteed.

[0109] In general, this technology successfully prepared high-quality, highly active collagen freeze-dried powder by optimizing the extraction and purification process, which has potential health and medical application value.

[0110] Comparative Example 1 removes acrylate derivatives. We can analyze the influence mechanism of removing acrylate derivatives on collagen extraction and performance in Comparative Example 1:

[0111] Reduced cross-linking ability:

[0112] Acrylate derivatives have active double bonds and can undergo cross-linking reactions with amino groups and other functional groups on collagen molecules. After removing this component, the degree of cross-linking between collagen molecules is reduced, resulting in a decrease in the structural stability of the extracted collagen. Water solubility deteriorates: Acrylic acid and its derivatives are hydrophilic and can increase the water solubility of collagen. After removal, the water solubility of the extracted collagen decreases, which is not conducive to the subsequent separation and purification process. Reduced extraction efficiency: Acrylate derivatives play an auxiliary role in the collagen extraction process, such as helping to destroy the cross-linking between collagen fibers. After removal, the extraction efficiency decreases and the product yield decreases. Affected biological activity: Some acrylate derivatives help maintain the biological activity of collagen. After removal, the biological activity of the extracted collagen decreases, such as the ACE inhibitory activity decreases. Decreased purity: Acrylate derivatives play a selective role in the extraction process and help to improve the purity of the target product. After removal, the impurity removal effect deteriorates and the purity of the final product decreases. Loss of functional diversity: Acrylate grafting can give collagen new functional properties. After removal, the product loses these potential functional advantages, limiting its application range. In summary, the removal of acrylate derivatives leads to negative impacts on collagen extraction efficiency, purity, water solubility, stability and biological activity, which is ultimately reflected in the significant decrease in product yield and performance. This shows that acrylate derivatives play an important role in the preparation of the collagen freeze-dried powder and are one of the key components in the process.

[0113] Comparative Example 2 replaces weak base with sodium hydroxide, which has a significant impact on the extraction process of collagen and the quality of the final product. The following is an analysis of the main mechanism of this replacement: Overhydrolysis: Sodium hydroxide is a strong base and has a stronger alkalinity than weak bases (such as sodium bicarbonate). A strong alkaline environment can lead to excessive hydrolysis of collagen peptide bonds and destroy the triple helix structure of collagen. This structural damage can reduce the biological activity and functional properties of collagen. Amino acid destruction: Under strong alkaline conditions, some hydroxyl- and sulfhydryl-containing amino acids (such as serine, threonine, and cysteine) are easily destroyed. This not only affects the nutritional value of collagen, but also changes its physicochemical properties. Racemization: Sodium hydroxide treatment causes racemization of certain amino acids to produce D-type amino acids. D-type amino acids are not only difficult to be used by the human body, but some are also toxic. Changes in extraction efficiency: Although sodium hydroxide improves the extraction efficiency of collagen in the short term, due to excessive hydrolysis, it eventually leads to the loss of effective ingredients and reduces the overall extraction efficiency. Impurity removal effect: Strong alkali treatment is more effective in removing certain impurities, but it also causes partial loss of target collagen. Increased difficulty in pH adjustment: After using a strong base, it will be more difficult to adjust the pH to neutral or weak acid in subsequent treatments, and more acid will be required for neutralization, which increases the process complexity and cost. Reduced product stability: Due to the destruction of the triple helix structure, the thermal stability and storage stability of the final product will be reduced. Changes in biocompatibility: Excessive hydrolysis and structural changes affect the biocompatibility of collagen, especially when used in the medical or cosmetic fields. Special attention should be paid. In summary, although replacing the weak base with sodium hydroxide improves the extraction efficiency in some aspects, it will generally have a negative impact on the structural integrity, biological activity and product quality of collagen. This explains why the product yield and purity in Comparative Example 2 are lower than those in the example using a weak base. In the process of collagen extraction, choosing a suitable alkaline reagent and controlling the reaction conditions are crucial to obtaining high-quality products.

[0114] Comparative Example 3 replaces the polyether derivative with ether. We can analyze the mechanism of the effect of replacing the polyether derivative with ether on the collagen extraction process: Difference in solubility: Polyether derivatives (such as polyether sulfone) have strong polarity and solubility, which can better dissolve and disperse collagen. Ether is a non-polar solvent and has poor solubility for collagen, resulting in reduced extraction efficiency. Selective extraction: Polyether derivatives have a certain selectivity for collagen, which helps to improve product purity. Ether lacks this selectivity and will extract other unwanted proteins and impurities at the same time. Protein conformation protection: Polyether derivatives help maintain the natural conformation of collagen, while ether as an organic solvent will cause partial denaturation of proteins and affect their biological activity. Stability of the extraction process: The high volatility of ether leads to unstable solvent concentration during the extraction process, affecting the consistency of the extraction effect. Polyether derivatives are relatively stable and can maintain more consistent extraction conditions. Safety considerations: The flammable and explosive properties of ether increase operational risks, while polyether derivatives are relatively safe and more suitable for industrial production. Difficulty of post-processing: The use of ether requires more complex post-processing steps to remove residual solvents, while polyether derivatives are easier to remove by simple methods (such as dialysis). Impact on product quality: Due to the combined effect of the above factors, the use of ether results in the final product yield, purity and biological activity being lower than those of the use of polyether derivatives. In summary, replacing the polyether derivatives with ether reduces the extraction effect of collagen in many aspects, which explains why the product yield and purity of Comparative Example 3 are significantly lower than those of the embodiment. This replacement not only affects the extraction efficiency, but also has a negative impact on product quality and production safety.

[0115] Comparative Example 4 does not use dopamine analogs, which will have a significant impact on the preparation process of collagen freeze-dried powder and the performance of the final product. The following is an analysis of the main mechanism of this change: Reduced cross-linking ability: Dopamine analogs have multiple functional groups and can form cross-links with collagen molecules. After removing dopamine analogs, the degree of cross-linking between collagen molecules is reduced, resulting in a decrease in the structural stability and mechanical strength of the final product. Weakened antioxidant performance: Dopamine and its analogs have strong antioxidant capacity. Not using dopamine analogs results in a significant decrease in the antioxidant performance of the product, making collagen more susceptible to oxidative damage. Changes in biological activity: Dopamine analogs form complexes with collagen to enhance its biological activity. After removing dopamine analogs, the ACE inhibitory activity of the product is reduced, which explains why the IC50 value of Comparative Example 4 (0.091 mg / mL) is higher than that of the embodiment (0.042-0.059 mg / mL). Solubility and dispersibility effects: Dopamine analogs help to improve the dispersibility and stability of collagen in solution. After removal, the solubility and dispersibility of the product in subsequent applications are reduced. Changes in surface properties: Dopamine analogs change the surface characteristics of collagen particles, affecting their interaction with other molecules. Not using dopamine analogs causes changes in the surface properties of the product, affecting its performance in applications. Purity impact: Although the removal of dopamine analogs slightly improves the purity of the product, it can be seen from Table 1 that the purity of Example 4 (81.4%) is still lower than that of the embodiment (98.2-99.2%). This shows that dopamine analogs play a positive role in improving product quality. Change in yield: The yield of Example 4 (80.1%) is lower than that of the embodiment (93.4-94.2%), indicating that dopamine analogs play an important role in improving product yield, by enhancing the stability of collagen or improving its extraction efficiency. In summary, not using dopamine analogs will lead to changes in the structural stability, antioxidant properties, biological activity, solubility, surface properties and other aspects of collagen freeze-dried powder, which is ultimately reflected in the significant reduction in product yield, purity and ACE inhibitory activity. This shows that dopamine analogs play an important role in the preparation of the collagen freeze-dried powder and are one of the key components in the process.

[0116] Comparative Example 5 replaces acetone with pure water. We can analyze the mechanism of the influence of replacing acetone with pure water on the collagen extraction process in Comparative Example 5: Difference in solubility: Acetone is an organic solvent that has good solubility for certain non-polar substances. As a polar solvent, pure water has poor solubility for non-polar substances. After replacing acetone with pure water, some non-polar impurities cannot be effectively removed, thus affecting the purity of the final product. Dehydration effect: Acetone has a strong dehydration ability and can quickly remove water from collagen. Pure water cannot play this dehydration role, which will cause the final product to have a high water content, which is not conducive to drying and preservation. Protein conformation effect: Acetone as an organic solvent will partially change the higher-order structure of collagen, while pure water can better maintain the natural conformation of the protein. However, this conformational protection will make some impurities more difficult to remove. Freeze-drying effect: The samples treated with acetone will form more porous structures during the freeze-drying process, which is conducive to subsequent rehydration. The freeze-drying effect of the samples treated with pure water is not as ideal as that of acetone. Purity influence: From the data in Table 1, it can be seen that the product purity of Comparative Example 5 (76.5%) is significantly lower than that of the embodiment using acetone (98.2-99.2%). This shows that the use of pure water instead of acetone leads to a significant decrease in the purity of the final product. Yield change: Although the yield of Comparative Example 5 (82.5%) is higher than that of other comparative examples, it is still lower than that of the embodiment (93.4-94.2%). This shows that the use of pure water will lead to the loss of some effective ingredients. Biological activity: From Table 2, it can be seen that the ACE inhibitory activity of Comparative Example 5 (IC50 value is 0.132 mg / mL) is significantly lower than that of the embodiment (0.042-0.059 mg / mL). This shows that the use of pure water instead of acetone will lead to a decrease in the biological activity of collagen. In summary, although replacing acetone with pure water has certain advantages in some aspects (such as protecting protein conformation), it will generally lead to a decrease in product purity, yield and biological activity. This is mainly because pure water lacks the dehydration ability and solubility of acetone for non-polar substances, and cannot effectively remove certain impurities and moisture. Therefore, in the preparation process of the collagen freeze-dried powder, using acetone as the solvent for the final processing step is a better choice.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing freeze-dried collagen powder, characterized in that: The following steps are included by weight: (1) Crude extraction: First, 0.1-0.5 parts of an emulsifier and 30-50 parts of fresh fish skin are added to a feed tank; then, 100-160 parts of deionized water are added to the feed tank, mixed and stirred, and heated in a water bath to 60-80° C. and kept constant for 2-6 hours; then, 15-20 parts of an acrylate derivative and 1-3 parts of a polymerization inhibitor are added to the feed tank, and mechanical stirring and emulsification are performed for 15-30 minutes; finally, 10-20 parts of a stabilizer are added, reacted at 35-50° C. for 8-12 hours, and ultrasonicated for 2-4 hours to obtain a suspension, and then 10-20 parts of the suspension are dispersed in 100-120 parts of anhydrous ethanol, and then the ethanol is evaporated at 50-70° C. for 4-6 hours to obtain a crude extraction product; (2) Fine extraction: 4-7 parts of weak base, 1-2 parts of dry chloride and 1-2 parts of buffer are provided and mixed with the crude extraction product obtained in step (1), and homogenized; then, the mixture is transferred to a sealed container and subjected to microwave treatment for 20-40 min under a nitrogen atmosphere, wherein the microwave power is 200-400 W, and the solid product is collected and rinsed with acid, then rinsed with deionized water and dried to obtain a fine extraction product; (3) Freeze-dried product: Add 2-5 parts of the crude extracted product obtained in step (2) to 20-40 parts of the polyether derivative, then ultrasonically treat to form a suspension, then add 1-3 parts of dopamine analogs, stir evenly, add ammonium salt solution dropwise at a uniform rate, stir and react at 40-60° C. for 2-3 hours, then centrifuge at 120,000-160,000 rpm for 20-30 minutes to collect the organic phase, add twice the volume of 4° C. acetone, ultrasonically mix, and place in an oven for drying to obtain a dry powder.

2. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: The emulsifier in step (1) is one of glyceryl monostearate, sucrose fatty acid ester, soybean lecithin, sorbitan monostearate, and polyoxyethylene sorbitan fatty acid ester; the acrylate derivative is one of aminoacrylate, 3-aminoacrylate methyl ester, and aminoacrylate dodecyl ester amide; the inhibitor is one of nano titanium dioxide, nano magnesium oxide, and nano sodium chloride; and the stabilizer is one of potassium sulfate, dibutyltin dilaurate, and dioctyltin thioglycolate.

3. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: The rotation speed of the mixing and stirring in step (1) is 300 rpm; the rotation speed of the mechanical stirring is 600 rpm; the ultrasonic method is water bath ultrasonication, the power of the ultrasonication is 250-350 W, and the temperature of the ultrasonication is below 15°C.

4. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: In step (2), the weak base is one of sodium bicarbonate and ammonium hydroxide; the chloride is one of magnesium chloride, zinc chloride and calcium chloride; the buffer is one of PBS, Tris-HCl and sodium acetate; the acid rinse is hydrochloric acid rinse, wherein the concentration of hydrochloric acid is 1 mM.

5. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: The pressure of the homogenization treatment in step (2) is 1MPa-3MPa, the time of the homogenization treatment is 2h-4h; the flow rate of the nitrogen is 100mL / min-200mL / min; and the temperature of the drying is 40-60°C.

6. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: In step (3), the polyether derivative is one of polyetheretherketone, polyetherimide, polyethersulfone, and polyethersulfoneetherketone; the dopamine analog is one of dopamine hydrochloride, dopamine-3-phosphate, and mercaptopolyethylene glycol dopamine; the ammonium salt solution is one of ammonium chloride solution, ammonium carbonate solution, and ammonium phosphate, and the concentration of the ammonium salt solution is 10mM.

7. The method for preparing the freeze-dried collagen powder according to claim 1, characterized in that: The ultrasonic method in step (3) is water bath ultrasonication, and the ultrasonic power is 80-250W; the stirring speed is 180rpm; the uniform dripping rate is 0.1-0.3 parts / min, and the total amount of the uniformly dripped solution is 4-8 parts; the ultrasonic mixing temperature is 40-50°C, and the ultrasonic mixing power is 100-300W; the oven drying temperature is 45-60°C.

8. A dry powder prepared by the method for preparing the lyophilized collagen powder according to any one of claims 1 to 7.

9. Use of the collagen freeze-dried powder according to any one of claims 1 to 7 in preparing an antioxidant preparation.

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