A preparation method of antioxidant sturgeon protein peptide

Abstract

The present invention discloses a preparation method of antioxidant sturgeon protein peptides, comprising the following steps: S1, extraction of crude sturgeon skin protein solution; S2, extraction of crude sturgeon swim bladder protein; S3, extraction of crude sturgeon meat protein solution; S4, mixed enzymatic hydrolysis; S5, subjecting the obtained enzymatic hydrolysis product to enzyme inactivation treatment first to obtain an enzymatic hydrolysis solution, and then sequentially ultrafiltering and vacuum drying the enzymatic hydrolysis solution to obtain antioxidant sturgeon protein peptides. The present invention mixes the proteins extracted from different parts of sturgeon in a specific ratio, and then performs enzymatic hydrolysis. The neutral protease and papain added can act synergistically to more comprehensively hydrolyze proteins, generating small peptides with specific functions. At the same time, glutathione added during enzymatic hydrolysis can protect the sulfhydryl groups of the enzyme and maintain the enzyme activity, so that the finally obtained sturgeon protein peptides have good antioxidant properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquatic product processing, and particularly relates to a preparation method of antioxidant sturgeon protein peptides. Background Art

[0002] Sturgeon, also known as Acipenser, is the largest and longest-lived fish among freshwater fish, and is also one of the oldest and most primitive groups of fish on the earth. It is a treasure all over, with high economic value and research value. The sturgeon meat is thick and boneless, and its meat quality is better than that of salmon, with high nutritional value. The protein content in its fish roe is quite high, and the sugar content is low, with high edible value. China's sturgeon aquaculture industry has achieved remarkable results in artificial cultivation and breeding technology, and the aquaculture scale and output of sturgeon have been increasing year by year. The main wastes after deep processing of sturgeon - fish viscera, fish bones and fish skins have relatively high contents of protein, chondroitin sulfate and anti-cancer factors, and have certain nutritional value, so they can be used as raw materials for extracting natural active substances.

[0003] The main components of sturgeon include essential amino acids, vitamins, minerals, phosphorus, potassium, etc. Among them, the protein content is as high as 18%, and the protein content in sturgeon roe can be as high as 29%. Sturgeon meat contains a variety of unsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish rich in unsaturated fatty acids are crucial for human health. Eating sturgeon has a positive regulatory effect on human blood pressure, metabolic process, skin regeneration, etc. The abdomen, nose, tendons, bones, etc. of sturgeon can be cooked into special Chinese dishes. Moreover, the chondroitin sulfate content in its cartilage is high, and eating its bone products has the effects of enhancing immunity and anti-aging; its fish skin can be made into glue and is also a high-grade leather raw material.

[0004] Bioactive peptides are a class of proteolytic intermediate products with a relative molecular mass of less than 6 kDa formed by dehydration condensation of 2 - 20 amino acids, and have biological functions such as enhancing human immunity, antioxidant, blood pressure lowering and bacteriostasis. Bioactive peptides have a wide range of sources and can generally be divided into plant peptides and animal peptides. Animal peptides are composed of natural carnosine, marine bioactive peptides, dairy peptides, etc. Marine organisms are more complex and special than other organisms, which makes their protein peptides prone to produce effects different from those of other bioactive peptides.

[0005] At present, the common techniques for extracting sturgeon skin collagen and collagen peptides are: acid extraction method, alkali extraction method, salt solution extraction method, heat extraction method and enzyme extraction method. However, there are the following deficiencies: The extraction process is complex: Traditional extraction methods usually require multiple processes, including pretreatment, enzymatic hydrolysis, extraction and other steps, with a long process flow and complex operations. The extraction efficiency is low: The extraction methods in the existing technologies often have low efficiency and may damage the activity of proteins, affecting the efficacy of the final product. The process conditions are harsh: The current preparation methods usually need to be carried out under specific conditions such as temperature and pH value, with high requirements for equipment and operations, increasing the production cost. The utilization rate of raw materials is insufficient: The comprehensive utilization degree of sturgeon resources in the existing technologies is not high, and often only focuses on the extraction of a specific part, resulting in resource waste. Chinese Patent Application CN109880872A discloses a method for extracting collagen peptides from sturgeon, including the following steps: S1, obtaining raw materials; S2, pulverizing; S3, pretreatment; S4, extraction; S5, enzymatic hydrolysis;

[0006] S6, decolorizing and filtering; S7, membrane filtration; S8, vacuum concentration; S9, centrifugal spray drying. After the sturgeon protein is moderately hydrolyzed, its water solubility can usually be significantly improved, enabling the full and more reasonable utilization of high-quality protein resources. By hydrolyzing proteins with proteases, the degree of hydrolysis is high, the protein extraction utilization rate is high, the process conditions are stable and easy to control. In the processing, proteases are mainly used to enzymatically hydrolyze substrates to obtain products or improve product quality. The enzyme technology is used to enzymatically hydrolyze sturgeon collagen peptides, and then a nanofiltration membrane device is used to separate and purify the collagen peptides, which is suitable for large-scale production in factories. However, the extraction process of this invention is complex, and it is impossible to see what effects the extracted collagen peptides have from its application text.

[0007] Therefore, it has important practical significance to develop a mild preparation method that can maintain the activity of sturgeon protein peptides in this field. At the same time, improving the comprehensive utilization rate of raw materials and realizing the sustainable utilization of sturgeon resources are also problems that need to be solved urgently. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a preparation method of antioxidant sturgeon protein peptides.

[0009] To achieve the above purpose, the present invention provides the following technical solutions:

[0010] A preparation method of antioxidant sturgeon protein peptides, including the following steps:

[0011] S1. Extraction of crude sturgeon skin protein solution: Wash the sturgeon skin and cut it into pieces. Then soak it in a sodium chloride solution, wash it after soaking, and then extract it in petroleum ether. After extraction, defatted sturgeon skin is obtained. Add the defatted sturgeon skin to a tartaric acid solution for swelling, and then add tea polyphenols and pepsin for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme and filter to obtain the crude sturgeon skin protein solution.

[0012] S2. Extraction of crude sturgeon swim bladder protein: Wash the sturgeon swim bladder and cut it into pieces. Then soak it in a sodium hydroxide solution, wash it until neutral after soaking, and then add it to an acetic acid solution for stirring extraction. After extraction, centrifuge, collect the supernatant, add sodium chloride for salting out, centrifuge after standing, and dry the precipitate to obtain the crude sturgeon swim bladder protein.

[0013] S3. Extraction of crude sturgeon meat protein solution: Wash the sturgeon meat, remove the surface connective tissue and fat, cut it into small pieces, wash it 2 - 3 times with distilled water to remove blood and impurities, then add it to a blender to make minced meat. Add the minced meat to distilled water, then add cysteine and pepsin for stirring enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme, centrifuge, and collect the supernatant to obtain the crude sturgeon meat protein solution.

[0014] S4. Mixed enzymatic hydrolysis: Mix the crude sturgeon skin protein solution in step S1, the crude sturgeon swim bladder protein in step S2, and the crude sturgeon meat protein solution in step S3 evenly to obtain a mixed solution. Then add glutathione, neutral protease, and papain for mixed enzymatic hydrolysis. After enzymatic hydrolysis, centrifuge and take the supernatant to obtain the enzymatic hydrolysis product.

[0015] S5. First, inactivate the enzyme of the obtained enzymatic hydrolysis product to obtain an enzymatic hydrolysis solution, and then sequentially ultrafilter and vacuum dry the enzymatic hydrolysis solution to obtain antioxidant sturgeon protein peptides.

[0016] Preferably, in step S1, the mass concentration of the sodium chloride solution is 4 - 6%, the soaking temperature is 5 - 10°C, and the time is 12 - 18 h; the extraction temperature is 4 - 8°C, and the time is 24 - 30 h.

[0017] Preferably, in step S1, the mass concentration of the tartaric acid solution is 2 - 4%, the material - liquid ratio of the defatted sturgeon skin to the tartaric acid solution is 1:10 - 15, the swelling temperature is 4 - 8°C, and the time is 18 - 24 h. The addition amount of tea polyphenols is 1 - 2% of the mass of the defatted sturgeon skin, the addition amount of pepsin is 0.5 - 1% of the mass of the defatted sturgeon skin, the enzymatic hydrolysis temperature is 30 - 35°C, and the time is 2 - 4 h.

[0018] In the present invention, the sturgeon skin is pretreated by soaking in a sodium chloride solution for cleaning and softening the fish skin tissue. Subsequently, petroleum ether is used as a degreasing agent to remove the fat components. Then, it is added to a tartaric acid solution to promote tissue swelling for facilitating enzymatic hydrolysis. Finally, enzymatic hydrolysis is carried out to extract the protein from the sturgeon skin. And during the enzymatic hydrolysis process, tea polyphenols are added. On the one hand, it has antioxidant effects and can protect proteins from oxidative damage. On the other hand, it can also promote the stability of proteases and maintain enzyme activity. This method combines physical and biochemical methods to complete the extraction process, improving the antioxidant property of the crude sturgeon skin protein solution.

[0019] Preferably, in step S2, the concentration of the sodium hydroxide solution is 0.1 - 0.2 mol / L, the temperature of the impregnation is 4 - 6 °C, and the time is 20 - 24 h.

[0020] Preferably, in step S2, the concentration of the acetic acid solution is 0.5 - 0.8 mol / L, the material - liquid ratio of the sturgeon swim bladder to the acetic acid solution is 1:10 - 15, the temperature of the stirring extraction is 4 - 6 °C, and the time is 36 - 42 h; sodium chloride is added to a concentration of 1.2 mol / L during salting - out.

[0021] In the present invention, during the treatment of the sturgeon swim bladder, the sodium hydroxide solution is used for tissue softening and collagen dissolution, the acetic acid solution aids in protein extraction, and sodium chloride for salting - out can precipitate and separate the target protein, thereby extracting the protein from the sturgeon swim bladder.

[0022] Preferably, in step S3, the mass ratio of the meat paste to distilled water is 1:5 - 8, the addition amount of cysteine is 2 - 3% of the mass of the meat paste, the addition amount of pepsin is 1 - 2% of the mass of the meat paste, the temperature of the stirring enzymatic hydrolysis is 30 - 35 °C, the time is 4 - 6 h, and the stirring rate is 80 - 120 r / min.

[0023] In the present invention, for extracting the protein from sturgeon meat by the enzymatic method, the added cysteine can act as a reducing agent to help break the disulfide bonds between protein molecules. At the same time, it can also protect the sulfhydryl groups of the enzyme, maintain enzyme activity, and improve the protein extraction efficiency.

[0024] Preferably, in step S4, the mass ratio of the crude sturgeon skin protein solution, the crude sturgeon swim bladder protein, and the crude sturgeon meat protein solution is 100 - 150:20 - 30:70 - 120.

[0025] Preferably, in step S4, the addition amount of glutathione is 0.2 - 0.4% of the mass of the mixed solution, the addition amount of neutral protease is 0.5 - 1% of the mass of the mixed solution, the addition amount of papain is 1 - 1.5% of the mass of the mixed solution, and the temperature of the mixed enzymatic hydrolysis is 45 - 50 °C, and the time is 1 - 2 h.

[0026] In the present invention, proteins extracted from different parts of sturgeon are mixed and enzymatically hydrolyzed. The neutral protease and papain added can act synergistically to more comprehensively hydrolyze proteins, generating small peptides with specific functions. At the same time, the glutathione added can protect the sulfhydryl groups of the enzymes, maintain the enzyme activity, and enable the finally obtained sturgeon protein peptides to have good antioxidant properties.

[0027] Preferably, in step S5, the enzyme inactivation is carried out in a boiling water bath for 10 - 15 min, and the ultrafiltration uses a membrane with a pore size of 3000D for ultrafiltration.

[0028] The present invention also protects an antioxidant sturgeon protein peptide prepared by the method as described above.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The preparation method of the antioxidant sturgeon protein peptide provided by the present invention mixes the proteins extracted from different parts of sturgeon in a specific proportion and then carries out enzymatic hydrolysis. The neutral protease and papain added can act synergistically to more comprehensively hydrolyze proteins, generating small peptides with specific functions. At the same time, the glutathione added during enzymatic hydrolysis can protect the sulfhydryl groups of the enzymes, maintain the enzyme activity, and enable the finally obtained sturgeon protein peptides to have good antioxidant properties.

[0031] (2) The preparation method of the antioxidant sturgeon protein peptide provided by the present invention pre-treats the sturgeon skin by soaking it in a sodium chloride solution for cleaning and softening the fish skin tissue. Subsequently, petroleum ether is used as a degreasing agent to remove the fat components. Then, it is added to a tartaric acid solution to promote tissue swelling for facilitating enzymatic hydrolysis. Finally, enzymatic hydrolysis is carried out to extract the protein from the sturgeon skin. And during the enzymatic hydrolysis process, tea polyphenols are added. On the one hand, it has antioxidant effects and can protect proteins from oxidative damage. On the other hand, it can also promote the stability of proteases and maintain the enzyme activity. This method combines physical and biochemical methods to complete the extraction process, improving the antioxidant property of the crude sturgeon skin protein solution; the protein in sturgeon meat is extracted by an enzymatic method. The cysteine added can act as a reducing agent to help break the disulfide bonds between protein molecules, and at the same time, it can also protect the sulfhydryl groups of the enzymes, maintain the enzyme activity, and improve the protein extraction efficiency. Specific Embodiments

[0032] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0033] In the present invention, the tea polyphenols are purchased from Lanzhou Wateles Biotech Co., Ltd. and are green tea extracts with an extraction specification of 10:1; the pepsin is purchased from Guangzhou Huayu Biotech Co., Ltd. with an enzyme activity of 50,000 U / g; the neutral protease is purchased from Hebei Jiuyu Biotech Co., Ltd. with an enzyme activity of 100,000 U / g; the papain is purchased from Guangzhou Huayu Biotech Co., Ltd. with an enzyme activity of 100,000 U / g.

[0034] Example 1

[0035] A preparation method of antioxidant sturgeon protein peptide, comprising the following steps:

[0036] S1. Extraction of sturgeon skin crude protein solution: Wash the sturgeon skin and cut it into small pieces of 5 mm×5 mm, then add it to a sodium chloride solution with a mass concentration of 5% and soak it at a temperature of 8°C for 15 h. After soaking, wash it with distilled water, then add it to petroleum ether and extract it at 6°C for 27 h. After extraction, obtain defatted sturgeon skin; Add 100 g of defatted sturgeon skin to 1.3 L of a tartaric acid solution with a mass concentration of 3% and swell it at 6°C for 20 h, then add 1.5 g of tea polyphenols and 0.8 g of pepsin, and enzymatically hydrolyze it at 33°C for 3 h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10 min, filter, centrifuge the filtrate at 4000 r / min for 10 min, and take the supernatant to obtain sturgeon skin crude protein solution;

[0037] S2. Extraction of sturgeon swim bladder crude protein: Wash the sturgeon swim bladder and cut it into small pieces of 5 mm×5 mm, then add it to a sodium hydroxide solution with a concentration of 0.15 mol / L and impregnate it at a temperature of 5°C for 22 h. After impregnation, wash it with distilled water until neutral, then add 100 g of sturgeon swim bladder to 1.3 L of an acetic acid solution with a concentration of 0.7 mol / L and stir and extract it at 5°C for 38 h. After extraction, centrifuge it at 4000 r / min for 10 min, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand and then centrifuge it at 4000 r / min for 10 min, and dry the precipitate to obtain sturgeon swim bladder crude protein;

[0038] S3. Extraction of sturgeon meat crude protein solution: Wash the sturgeon meat, remove the surface connective tissue and fat, then cut it into small pieces, wash it with distilled water 2-3 times to remove blood and impurities, then add it to a blender to make minced meat. Add 100 g of minced meat to 700 g of distilled water, then add 2.5 g of cysteine and 1.5 g of pepsin, and enzymatically hydrolyze it at a stirring rate of 100 r / min and a temperature of 33°C for 5 h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10 min, centrifuge it at 4000 r / min for 10 min, and collect the supernatant to obtain sturgeon meat crude protein solution;

[0039] S4. Mixed enzymatic hydrolysis: Mix 130 g of sturgeon skin crude protein solution in step S1, 25 g of sturgeon swim bladder crude protein in step S2, and 100 g of sturgeon meat crude protein solution in step S3 evenly to obtain a mixed solution. Then add 0.765 g of glutathione, 0.765 g of neutral protease, and 3.315 g of papain, and perform mixed enzymatic hydrolysis at 48 °C for 1.5 h. After the enzymatic hydrolysis is completed, centrifuge at 4000 r / min for 10 min, and take the supernatant to obtain an enzymatic hydrolysis product;

[0040] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000 D and perform vacuum drying to obtain antioxidant sturgeon protein peptides.

[0041] Example 2

[0042] A preparation method of antioxidant sturgeon protein peptides, comprising the following steps:

[0043] S1. Extraction of sturgeon skin crude protein solution: Wash the sturgeon skin and cut it into small pieces of 5 mm × 5 mm. Then add it to a sodium chloride solution with a mass concentration of 4% and soak it at a temperature of 5 °C for 18 h. After soaking, wash it with distilled water, then add it to petroleum ether and perform extraction at 4 °C for 30 h. After the extraction is completed, obtain defatted sturgeon skin; Add 100 g of defatted sturgeon skin to 1 L of a tartaric acid solution with a mass concentration of 4% and swell it at 4 °C for 24 h. Then add 1 g of tea polyphenols and 0.5 g of pepsin, and perform enzymatic hydrolysis at 30 °C for 4 h. After the enzymatic hydrolysis is completed, inactivate the enzyme in boiling water for 10 min, filter, centrifuge the filtrate at 4000 r / min for 10 min, and take the supernatant to obtain sturgeon skin crude protein solution;

[0044] S2. Extraction of sturgeon swim bladder crude protein: Wash the sturgeon swim bladder and cut it into small pieces of 5 mm × 5 mm. Then add it to a sodium hydroxide solution with a concentration of 0.1 mol / L and impregnate it at a temperature of 4 °C for 24 h. After impregnation, wash it with distilled water until neutral. Then add 100 g of sturgeon swim bladder to 1 L of an acetic acid solution with a concentration of 0.5 mol / L and stir and extract it at 4 °C for 42 h. After the extraction is completed, centrifuge at 4000 r / min for 10 min, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand, and then centrifuge at 4000 r / min for 10 min. Dry the precipitate to obtain sturgeon swim bladder crude protein;

[0045] S3. Extraction of crude sturgeon meat protein solution: Wash the sturgeon meat, cut it into small pieces after removing the surface connective tissue and fat, wash it 2 - 3 times with distilled water to remove blood and impurities, then add it to a blender to make minced meat. Add 100 g of minced meat to 500 g of distilled water, then add 2 g of cysteine and 1 g of pepsin, and enzymatically hydrolyze at a stirring rate of 80 r / min and a temperature of 30 °C for 6 h. After the enzymatic hydrolysis is completed, inactivate the enzyme in boiling water for 10 min, centrifuge at 4000 r / min for 10 min, and collect the supernatant to obtain the crude sturgeon meat protein solution;

[0046] S4. Mixed enzymatic hydrolysis: Mix 100 g of the crude sturgeon skin protein solution in step S1, 20 g of the crude sturgeon swim bladder protein in step S2, and 70 g of the crude sturgeon meat protein solution in step S3 evenly to obtain a mixed solution. Then add 0.38 g of glutathione, 0.38 g of neutral protease, and 1.9 g of papain, and perform mixed enzymatic hydrolysis at 45 °C for 2 h. After the enzymatic hydrolysis is completed, centrifuge at 4000 r / min for 10 min, and take the supernatant to obtain the enzymatic hydrolysis product;

[0047] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000 D and vacuum dry it to obtain the antioxidant sturgeon protein peptide.

[0048] Example 3

[0049] A preparation method of antioxidant sturgeon protein peptide, comprising the following steps:

[0050] S1. Extraction of crude sturgeon skin protein solution: Wash the sturgeon skin and cut it into small pieces of 5 mm × 5 mm, then add it to a sodium chloride solution with a mass concentration of 6% and soak it at a temperature of 10 °C for 12 h. After soaking, wash it with distilled water, then add it to petroleum ether and extract at 8 °C for 24 h. After the extraction is completed, obtain defatted sturgeon skin; Add 100 g of defatted sturgeon skin to 1.5 L of a tartaric acid solution with a mass concentration of 2%, swell it at 8 °C for 18 h, then add 2 g of tea polyphenols and 1 g of pepsin, and enzymatically hydrolyze at 35 °C for 2 h. After the enzymatic hydrolysis is completed, inactivate the enzyme in boiling water for 10 min, filter, centrifuge the filtrate at 4000 r / min for 10 min, and take the supernatant to obtain the crude sturgeon skin protein solution;

[0051] S2. Extraction of crude sturgeon swim bladder protein: Wash the sturgeon swim bladder and cut it into small pieces of 5 mm×5 mm. Then immerse it in a sodium hydroxide solution with a concentration of 0.2 mol / L at a temperature of 6 °C for 20 h. After immersion, wash it with distilled water until neutral. Then add 100 g of sturgeon swim bladder to 1.5 L of acetic acid solution with a concentration of 0.8 mol / L and stir and extract at 6 °C for 36 h. After extraction, centrifuge at 4000 r / min for 10 min, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand and then centrifuge at 4000 r / min for 10 min, and dry the precipitate to obtain crude sturgeon swim bladder protein;

[0052] S3. Extraction of crude sturgeon meat protein solution: Wash the sturgeon meat, remove the surface connective tissue and fat and cut it into small pieces, wash it with distilled water 2 - 3 times to remove blood and impurities, then add it to a blender to make minced meat. Add 100 g of minced meat to 800 g of distilled water, then add 3 g of cysteine and 2 g of pepsin, and enzymatically hydrolyze at a stirring rate of 120 r / min and a temperature of 35 °C for 4 h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10 min, centrifuge at 4000 r / min for 10 min, and collect the supernatant to obtain the crude sturgeon meat protein solution;

[0053] S4. Mixed enzymatic hydrolysis: Mix 150 g of crude sturgeon skin protein solution in step S1, 30 g of crude sturgeon swim bladder protein in step S2, and 120 g of crude sturgeon meat protein solution in step S3 evenly to obtain a mixed solution. Then add 1.2 g of glutathione, 1.2 g of neutral protease, and 4.5 g of papain, and perform mixed enzymatic hydrolysis at 50 °C for 1 h. After enzymatic hydrolysis, centrifuge at 4000 r / min for 10 min, and take the supernatant to obtain the enzymatic hydrolysis product;

[0054] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 15 min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000 D and vacuum dry it to obtain antioxidant sturgeon protein peptides.

[0055] Comparative Example 1

[0056] A preparation method of antioxidant sturgeon protein peptides, comprising the following steps:

[0057] S1. Extraction of crude protein from sturgeon swim bladder: Wash the sturgeon swim bladder and cut it into small pieces of 5mm×5mm. Then immerse it in a sodium hydroxide solution with a concentration of 0.15mol / L at a temperature of 5°C for 22h. After immersion, wash it with distilled water until neutral. Then add 100g of sturgeon swim bladder to 1.3L of acetic acid solution with a concentration of 0.7mol / L and stir and extract at 5°C for 38h. After extraction, centrifuge at 4000r / min for 10min, collect the supernatant, add sodium chloride to a concentration of 1.2mol / L for salting out, let it stand and then centrifuge at 4000r / min for 10min, and dry the precipitate to obtain crude protein from sturgeon swim bladder;

[0058] S2. Extraction of crude protein solution from sturgeon meat: Wash the sturgeon meat, remove the surface connective tissue and fat, cut it into small pieces, wash it 2 - 3 times with distilled water to remove blood and impurities, then add it to a blender to make minced meat. Add 100g of minced meat to 700g of distilled water, then add 2.5g of cysteine and 1.5g of pepsin, and enzymatically hydrolyze at a stirring rate of 100r / min and a temperature of 33°C for 5h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, and collect the supernatant to obtain the crude protein solution of sturgeon meat;

[0059] S3. Mixed enzymatic hydrolysis: Mix 25g of crude protein from sturgeon swim bladder in step S1 and 100g of crude protein solution from sturgeon meat in step S2 evenly to obtain a mixed solution. Then add 0.375g of glutathione, 0.375g of neutral protease, and 1.625g of papain, and perform mixed enzymatic hydrolysis at 48°C for 1.5h. After enzymatic hydrolysis, centrifuge at 4000r / min for 10min, and take the supernatant to obtain the enzymatic hydrolysis product;

[0060] S4. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000D and vacuum dry it to obtain antioxidant sturgeon protein peptides.

[0061] Compared with Example 1, the crude protein solution of sturgeon skin was not added in this comparative example.

[0062] Comparative Example 2

[0063] A preparation method of antioxidant sturgeon protein peptides, comprising the following steps:

[0064] S1. Extraction of crude sturgeon skin protein solution: Wash the sturgeon skin and cut it into small pieces of 5mm×5mm. Then soak it in a sodium chloride solution with a mass concentration of 5%, at a soaking temperature of 8°C for 15h. After soaking, wash it with distilled water, then add it to petroleum ether and extract at 6°C for 27h to obtain defatted sturgeon skin. Add 100g of defatted sturgeon skin to 1.3L of tartaric acid solution with a mass concentration of 3%, swell at 6°C for 20h, then add 1.5g of tea polyphenols and 0.8g of pepsin, enzymatically hydrolyze at 33°C for 3h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10min, filter, centrifuge the filtrate at 4000r / min for 10min, and take the supernatant to obtain the crude sturgeon skin protein solution;

[0065] S2. Extraction of crude sturgeon swim bladder protein: Wash the sturgeon swim bladder and cut it into small pieces of 5mm×5mm. Then soak it in a sodium hydroxide solution with a concentration of 0.15mol / L, at a soaking temperature of 5°C for 22h. After soaking, wash it with distilled water until neutral. Then add 100g of sturgeon swim bladder to 1.3L of acetic acid solution with a concentration of 0.7mol / L, stir and extract at 5°C for 38h. After extraction, centrifuge at 4000r / min for 10min, collect the supernatant, add sodium chloride to a concentration of 1.2mol / L for salting out, let it stand and then centrifuge at 4000r / min for 10min, and dry the precipitate to obtain the crude sturgeon swim bladder protein;

[0066] S3. Mixed enzymatic hydrolysis: Mix 130g of the crude sturgeon skin protein solution in step S1 and 25g of the crude sturgeon swim bladder protein in step S2 evenly to obtain a mixed solution. Then add 0.465g of glutathione, 0.465g of neutral protease, and 2.015g of papain, and perform mixed enzymatic hydrolysis at 48°C for 1.5h. After enzymatic hydrolysis, centrifuge at 4000r / min for 10min, and take the supernatant to obtain the enzymatic hydrolysis product;

[0067] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000D and perform vacuum drying to obtain the antioxidant sturgeon protein peptide.

[0068] Compared with Example 1, the crude sturgeon meat protein solution was not added in this comparative example.

[0069] Comparative Example 3

[0070] A preparation method of antioxidant sturgeon protein peptide, comprising the following steps:

[0071] S1. Extraction of crude protein from sturgeon skin: Wash the sturgeon skin and cut it into small pieces of 5mm×5mm. Then soak it in a sodium chloride solution with a mass concentration of 5%, at a soaking temperature of 8°C for 15h. After soaking, wash it with distilled water, then add it to petroleum ether and extract at 6°C for 27h to obtain defatted sturgeon skin; Add 100g of defatted sturgeon skin to 1.3L of a tartaric acid solution with a mass concentration of 3%, swell at 6°C for 20h, then add 0.8g of pepsin and enzymatically hydrolyze at 33°C for 3h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10min, filter, centrifuge the filtrate at 4000r / min for 10min, and take the supernatant to obtain the crude protein solution of sturgeon skin;

[0072] S2. Extraction of crude protein from sturgeon swim bladder: Wash the sturgeon swim bladder and cut it into small pieces of 5mm×5mm. Then soak it in a sodium hydroxide solution with a concentration of 0.15mol / L, at a soaking temperature of 5°C for 22h. After soaking, wash it with distilled water until neutral. Then add 100g of sturgeon swim bladder to 1.3L of an acetic acid solution with a concentration of 0.7mol / L, stir and extract at 5°C for 38h. After extraction, centrifuge at 4000r / min for 10min, collect the supernatant, add sodium chloride to a concentration of 1.2mol / L for salting out, let it stand and then centrifuge at 4000r / min for 10min, and dry the precipitate to obtain the crude protein of sturgeon swim bladder;

[0073] S3. Extraction of crude protein solution from sturgeon meat: Wash the sturgeon meat, remove the surface connective tissue and fat, then cut it into small pieces, wash it with distilled water 2 - 3 times to remove blood and impurities, then add it to a blender to make minced meat. Add 100g of minced meat to 700g of distilled water, then add 1.5g of pepsin, and enzymatically hydrolyze at a stirring rate of 100r / min and a temperature of 33°C for 5h. After enzymatic hydrolysis, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, and collect the supernatant to obtain the crude protein solution of sturgeon meat;

[0074] S4. Mixed enzymatic hydrolysis: Mix 130g of the crude protein solution of sturgeon skin in step S1, 25g of the crude protein of sturgeon swim bladder in step S2, and 100g of the crude protein solution of sturgeon meat in step S3 evenly to obtain a mixed solution. Then add 0.765g of neutral protease and 3.315g of papain, and perform mixed enzymatic hydrolysis at 48°C for 1.5h. After enzymatic hydrolysis, centrifuge at 4000r / min for 10min, and take the supernatant to obtain the enzymatic hydrolysis product;

[0075] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10min to obtain an enzymatic hydrolysis solution, and then ultrafilter the enzymatic hydrolysis solution through a membrane with a pore size of 3000D and perform vacuum drying to obtain the antioxidant sturgeon protein peptide.

[0076] Compared with Example 1, in this comparative example, tea polyphenols were not added during the preparation of the crude sturgeon skin protein solution, cysteine was not added during the preparation of the crude sturgeon meat protein solution, and glutathione was not added during the mixed enzymatic hydrolysis.

[0077] The antioxidant properties of the sturgeon protein peptides prepared in Examples 1-3 and Comparative Examples 1-3 were tested as follows:

[0078] (1) Determination of DPPH free radical scavenging ability

[0079] The dried sturgeon protein peptides in Examples 1-3 and Comparative Examples 1-3 were respectively dissolved in 4 mL of distilled water to prepare sturgeon protein peptide solutions with a concentration of 4 mg / mL; 1 mL of an ethanol solution of DPPH free radicals with a concentration of 40 μmol / L was added to 4 mL of distilled water and each of the above sturgeon protein peptide solution samples, and they were left standing at 25 °C for 30 min. The absorbance values Ablank and Asample were measured at 517 nm respectively. The calculation formula for the DPPH free radical scavenging ability is:

[0080] DPPH free radical scavenging ability (%) = (1 - A 样品 / A 空白 ) × 100

[0081] (2) Determination of ABTS free radical scavenging ability:

[0082] The dried sturgeon protein peptides in Examples 1-3 and Comparative Examples 1-3 were respectively dissolved in 4 mL of distilled water to prepare sturgeon protein peptide solutions with a concentration of 4 mg / mL; the ABTS reaction solution was diluted with PBS buffer to make its absorbance value at 734 nm be 0.700 to prepare an ABTS assay solution. Take 4 mL of the ABTS assay solution, add 40 μL of the sample, and measure its absorbance value at 734 nm multiple times, and take the average value A. The calculation formula for the ABTS scavenging rate is:

[0083] ABTS scavenging rate (%) = (1 - A / 0.700) × 100

[0084] (3) Determination of the scavenging ability of hydroxyl radicals:

[0085] The dried sturgeon protein peptides of Examples 1-3 and Comparative Examples 1-3 were dissolved in 4 mL of distilled water to prepare sturgeon protein peptide solutions with a concentration of 4 mg / mL; the ability of the sturgeon protein peptide solution to remove ·OH was measured by Fenton reaction: 1 mL of 5 mmol / L o-phenanthroline ethanol solution was taken in a test tube and 2 mL of 0.2 mol / L phosphate buffer solution with a pH of 7.40 and 1 mL of sturgeon protein peptide solution were added in sequence and mixed thoroughly, then 1 mL of 5 mmol / L ferrous sulfate solution was added, and 1 mL of hydrogen peroxide was added after mixing. After 60 minutes of water bath at 37°C, the absorbance (A) was measured at a wavelength of 536 nm. 1 mL of distilled water was used to replace 1 mL of 0.1% hydrogen peroxide in the damaged tube, and 1 mL of sample solution was used to replace 1 mL of sample solvent in the damaged tube. The others were the same as the damaged tubes, and the absorbance of the undamaged tube (A) and the absorbance of the sample tube (A) were measured. 样 ), the calculation formula of OH removal rate is:

[0086] ·OH removal rate (%) = (A 样 -A 损 ) / (A 未 -A 损 )×100

[0087] The test results are shown in Table 1:

[0088] Table 1 Scavenging rate of sturgeon protein peptides of Examples 1-3 and Comparative Examples 1-3 on different free radicals

[0089]

[0090] It can be seen from Table 1 that the sturgeon protein peptides prepared in Examples 1-3 of the present invention have better antioxidant activity, indicating that the protein peptides mixed from sturgeon skin, sturgeon maw and sturgeon meat in the present invention have a synergistic effect and can effectively improve the overall antioxidant activity of the sturgeon protein peptides.

[0091] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an antioxidant sturgeon protein peptide, characterized in that: The following steps are involved: S1, washing and chopping the sturgeon skin, then soaking it in a sodium chloride solution, washing it after soaking, and then extracting it in petroleum ether to obtain defatted sturgeon skin; adding the defatted sturgeon skin to a tartaric acid solution for swelling, then adding tea polyphenols and pepsin for enzymolysis, killing the enzyme after the enzymolysis, filtering, and obtaining a crude protein liquid of sturgeon skin; S2, washing the sturgeon maw and chopping it, then adding it to a sodium hydroxide solution for immersion, washing it until it is neutral after the immersion is completed, then adding it to an acetic acid solution, stirring and extracting it, centrifuging it after the extraction is completed, collecting the supernatant, adding sodium chloride for salting out, standing it and then centrifuging it, and drying the precipitate to obtain sturgeon maw crude protein; S3, washing the sturgeon meat, removing the surface connective tissue and fat, chopping into small pieces, washing with distilled water 2-3 times to remove blood and impurities, then adding to a wall breaking machine to make minced meat, adding the minced meat to distilled water, then adding cysteine ​​and pepsin, stirring and enzymolysis, inactivating the enzyme after the enzymolysis is completed, centrifuging, collecting the supernatant, and obtaining a sturgeon meat crude protein liquid; S4, mixing the crude protein liquid of sturgeon skin in step S1, the crude protein of sturgeon maw in step S2, and the crude protein liquid of sturgeon meat in step S3 uniformly to obtain a mixed solution, then adding glutathione, neutral protease, and papain to perform mixed enzymolysis, centrifuging after the enzymolysis is completed, taking the supernatant, and obtaining an enzymolysis product; S5, first subjecting the obtained enzymatic hydrolysis product to enzyme inactivation treatment to obtain an enzymatic hydrolyzate, and then subjecting the enzymatic hydrolyzate to ultrafiltration and vacuum drying in sequence to obtain an antioxidant sturgeon protein peptide; Among them, the mass ratio of the sturgeon skin crude protein liquid, the sturgeon maw crude protein, and the sturgeon meat crude protein liquid in step S4 is 100-150:20-30:70-120; the amount of glutathione added is 0.2%-0.4% of the mass of the mixed solution, the amount of neutral protease added is 0.2%-0.4% of the mass of the mixed solution, the amount of papain added is 1%-1.5% of the mass of the mixed solution, and the temperature of the mixed enzymatic hydrolysis is 45-50°C, and the time is 1-2h.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the sodium chloride solution in step S1 is 4%-6%, the soaking temperature is 5-10°C, and the time is 12-18h; the extraction temperature is 4-8°C, and the time is 24-30h.

3. The preparation method according to claim 1, characterized in that: The mass concentration of the tartaric acid solution in step S1 is 2%-4%, the solid-liquid ratio of the defatted sturgeon skin to the tartaric acid solution is 1:10-15, the swelling temperature is 4-8°C, the time is 18-24h, the amount of tea polyphenols added is 1%-2% of the mass of the defatted sturgeon skin, the amount of pepsin added is 0.5%-1% of the mass of the defatted sturgeon skin, the enzymolysis temperature is 30-35°C, and the time is 2-4h.

4. The preparation method according to claim 1, characterized in that: The concentration of the sodium hydroxide solution in step S2 is 0.1-0.2 mol / L, the immersion temperature is 4-6° C., and the immersion time is 20-24 h.

5. The preparation method according to claim 1, characterized in that: The concentration of the acetic acid solution in step S2 is 0.5-0.8 mol / L, the solid-liquid ratio of the sturgeon swim bladder to the acetic acid solution is 1:10-15, the stirring extraction temperature is 4-6°C, and the time is 36-42h; sodium chloride is added to the salting out to a concentration of 1.2 mol / L.

6. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of the minced meat to distilled water is 1:5-8, the amount of cysteine ​​added is 2%-3% of the mass of the minced meat, the amount of pepsin added is 1%-2% of the mass of the minced meat, the temperature of the stirring enzymolysis is 30-35°C, the time is 4-6h, and the stirring rate is 80-120r / min.

7. The preparation method according to claim 1, characterized in that: The enzyme inactivation in step S5 is carried out in a boiling water bath for 10-15 minutes, and the ultrafiltration is carried out using a membrane ultrafiltration with a pore size of 3000D.

8. An antioxidant sturgeon protein peptide prepared by the method according to any one of claims 1 to 7.

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