Preparation method of efficient antioxidant sturgeon protein peptide

Abstract

The invention discloses a preparation method of efficient antioxidant sturgeon protein peptide. The preparation method comprises the following steps: S1, extracting a sturgeon skin crude protein solution; s2, extracting sturgeon swimming bladder crude protein; s3, extracting crude protein liquid of the sturgeon meat; s4, performing mixed enzymolysis; and S5, carrying out enzyme deactivation treatment on the obtained enzymatic hydrolysate to obtain enzymatic hydrolysate, and sequentially carrying out ultrafiltration and vacuum drying on the enzymatic hydrolysate to obtain the efficient antioxidant sturgeon protein peptide. Proteins extracted from different parts of sturgeons are mixed according to a specific proportion and then subjected to enzymolysis, the added neutral protease and papain can achieve a synergistic effect, protein is hydrolyzed more comprehensively, small molecule peptides with specific functions are generated, meanwhile, glutathione added during enzymolysis can protect sulfydryl of enzymes, and the enzymolysis effect of the sturgeons is improved. The enzyme activity is maintained, so that the finally obtained sturgeon protein peptide has good oxidation resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product processing, and particularly relates to a method for preparing a high-efficiency antioxidant sturgeon protein peptide. Background Art

[0002] Sturgeon, also known as sturgeon dragon, is the largest and longest-lived freshwater fish. It is also one of the oldest and most primitive groups of fish on earth. It is a treasure from head to toe and has high economic and research value. Sturgeon meat is thick and boneless, and its meat quality is better than salmon. It has high nutritional value. Its fish roe has a very high protein content and low sugar content, and has high edible value. my country's sturgeon farming industry has achieved remarkable results in artificial cultivation and breeding technology. The scale and output of sturgeon farming have increased year by year. The main waste after deep processing of sturgeon - fish viscera, fish bones, and fish skin - have high content of protein, chondroitin sulfate and anti-cancer factors, and have certain nutritional value. Therefore, 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 which the protein content is as high as 18%, and the protein content of 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 essential to 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 used to cook distinctive Chinese delicacies. In addition, the content of chondroitin sulfate in its cartilage is high. Eating its bone products has the effects of enhancing immunity and anti-aging. Its fish skin can be made into glue, which is also a high-grade leather raw material.

[0004] Biopeptides are a type of protein hydrolysis intermediates composed of 2-20 amino acids through dehydration condensation, with a relative molecular mass of less than 6kDa. They have biological functions such as enhancing human immunity, anti-oxidation, lowering blood pressure and inhibiting bacteria. Biopeptides come from a wide range of sources and can be roughly divided into plant peptides and animal peptides. Animal peptides are composed of natural carnosine, marine biological peptides, dairy peptides, etc. Marine organisms are more complex and special than other organisms, which makes their protein peptides easy to produce effects different from other biopeptides.

[0005] At present, the common technologies for extracting collagen and collagen peptides from sturgeon skin are: acid extraction, alkaline extraction, salt solution extraction, heat extraction and enzyme extraction. However, there are the following shortcomings: Complex extraction process: Traditional extraction methods usually require multiple steps, including pretreatment, enzymatic hydrolysis, extraction and other steps, and the process flow is lengthy and the operation is complicated. Low extraction efficiency: The extraction methods in the prior art are often inefficient and may damage the activity of the protein, affecting the efficacy of the final product. Harsh process conditions: The current preparation methods usually need to be carried out under specific temperature, pH value and other conditions, and have high requirements for equipment and operation, which increases production costs. Insufficient raw material utilization: The comprehensive utilization of sturgeon resources in the prior art is not high, and often only focuses on the extraction of a specific part, resulting in a waste of resources. Chinese patent application CN109880872A discloses a method for extracting collagen peptides from sturgeon, comprising the following steps: S1, raw material acquisition; S2, crushing; S3, pretreatment; S4, extraction; S5, enzymatic hydrolysis;

[0006] S6, decolorization, filtration; S7, membrane filtration; S8, vacuum concentration; S9, centrifugal spray drying. After moderate hydrolysis, the water solubility of sturgeon protein can usually be significantly improved, so that high-quality protein resources can be fully and more reasonably utilized. The protein is hydrolyzed by protease, with a high degree of hydrolysis, high protein extraction utilization rate, stable process conditions, and easy control. In processing, the protease is mainly used to hydrolyze the substrate to obtain the product or improve the product quality. The enzyme technology is used to hydrolyze the sturgeon collagen peptide, and the nanofiltration membrane equipment used later is used to separate and purify the collagen peptide, which is suitable for large-scale production in factories. However, the extraction process of the invention is complicated, and it is not clear from its application text what effect the extracted collagen peptide has.

[0007] Therefore, it is of great practical significance to develop an efficient, 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 urgent issues to be solved. Summary of the invention

[0008] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing a highly effective antioxidant sturgeon protein peptide.

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

[0010] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

[0011] S1. Extraction of crude protein liquid from sturgeon skin: 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 into a tartaric acid solution to swell it, then adding tea polyphenols and pepsin to perform enzymolysis, inactivating the enzyme after the enzymolysis, filtering it, and obtaining the crude protein liquid from sturgeon skin;

[0012] S2. Extraction of crude protein from sturgeon bladder: washing and chopping the sturgeon bladder, then immersing it in a sodium hydroxide solution, washing it until it is neutral, then adding it to an acetic acid solution, stirring and extracting it, centrifuging it after the extraction, collecting the supernatant, adding sodium chloride for salting out, standing it and then centrifuging it, and drying the precipitate to obtain crude protein from sturgeon bladder;

[0013] S3, extraction of crude protein liquid from sturgeon meat: wash the sturgeon meat, remove the surface connective tissue and fat, chop into small pieces, wash with distilled water 2-3 times to remove blood and impurities, then add to a wall breaking machine to make minced meat, add the minced meat to distilled water, then add cysteine ​​and pepsin, stir and enzymolyze, inactivate the enzyme after enzymolysis, centrifuge, collect the supernatant, and obtain the crude protein liquid from sturgeon meat;

[0014] S4, mixed enzymolysis: 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 are mixed evenly to obtain a mixed solution, and then glutathione, neutral protease, and papain are added to perform mixed enzymolysis, and after the enzymolysis is completed, the mixture is centrifuged, and the supernatant is taken to obtain an enzymolysis product;

[0015] S5. The obtained enzymatic hydrolysis product is first subjected to enzyme inactivation treatment to obtain an enzymatic hydrolysis solution, and then the enzymatic hydrolysis solution is subjected to ultrafiltration and vacuum drying in sequence to obtain a highly effective antioxidant sturgeon protein peptide.

[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 12-18h; the extraction temperature is 4-8°C, and the time is 24-30h.

[0017] Preferably, 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.

[0018] In the present invention, sturgeon skin is pretreated by soaking in a sodium chloride solution to clean and soften the fish skin tissue, and then petroleum ether is used as a degreasing agent to remove fat components, and then it is added to a tartaric acid solution to promote tissue swelling to facilitate enzymolysis, and finally enzymolysis is performed to extract protein from the sturgeon skin. In addition, tea polyphenols are added during the enzymolysis process. On the one hand, tea polyphenols have an antioxidant effect and can protect proteins from oxidative damage, and on the other hand, they can also promote the stability of proteases and maintain enzyme activity. The method combines physical and biochemical methods to complete the extraction process, thereby improving the antioxidant properties of sturgeon skin crude protein liquid.

[0019] Preferably, 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.

[0020] Preferably, the concentration of the acetic acid solution in step S2 is 0.5-0.8 mol / L, the solid-liquid ratio of the sturgeon maw to the acetic acid solution is 1:10-15, the temperature of the stirring extraction 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.

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

[0022] Preferably, 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.

[0023] In the present invention, protein in sturgeon meat is extracted by enzymatic method, and the added cysteine ​​can be used as a reducing agent to help break the disulfide bonds between protein molecules, and can also protect the sulfhydryl group of the enzyme, maintain the enzyme activity, and improve the protein extraction efficiency.

[0024] Preferably, 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.

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

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

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

[0028] The present invention also protects a high-efficiency 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 method for preparing the high-efficiency antioxidant sturgeon protein peptide provided by the present invention comprises mixing proteins extracted from different parts of sturgeon in a specific ratio and then performing enzymatic hydrolysis. The added neutral protease and papain can act synergistically to more comprehensively hydrolyze the protein to produce small molecule peptides with specific functions. At the same time, the glutathione added during enzymatic hydrolysis can protect the sulfhydryl group of the enzyme and maintain the enzyme activity, so that the finally obtained sturgeon protein peptide has good antioxidant properties.

[0031] (2) The preparation method of the high-efficiency antioxidant sturgeon protein peptide provided by the present invention comprises the following steps: pre-treating the sturgeon skin by soaking it in a sodium chloride solution to clean and soften the fish skin tissue, then using petroleum ether as a degreasing agent to remove the fat component, then adding it to a tartaric acid solution to promote tissue swelling to facilitate enzymatic hydrolysis, and finally performing enzymatic hydrolysis to extract the protein in the sturgeon skin. In addition, tea polyphenols are added during the enzymatic hydrolysis process. On the one hand, tea polyphenols have an antioxidant effect and can protect the protein from oxidative damage, and on the other hand, they can also promote the stability of proteases and maintain enzyme activity. This method combines physical and biochemical methods to complete the extraction process, thereby improving the antioxidant properties of the crude protein liquid from sturgeon skin. The protein in the sturgeon meat is extracted by enzymatic method, and the added cysteine ​​can be used as a reducing agent to help break the disulfide bonds between protein molecules, while also protecting the sulfhydryl groups of the enzyme, maintaining the enzyme activity, and improving the extraction efficiency of the protein. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0034] Example 1

[0035] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

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

[0037] S2. Extraction of crude protein from sturgeon bladder: clean the sturgeon bladder and chop it into small pieces of 5 mm×5 mm, then immerse it in a sodium hydroxide solution with a concentration of 0.15 mol / L, the immersion temperature is 5°C, the time is 22 hours, and after immersion, wash it with distilled water until it is neutral, then add 100 g of sturgeon bladder to 1.3 L of acetic acid solution with a concentration of 0.7 mol / L, stir and extract it at 5°C for 38 hours, centrifuge it at 4000 r / min for 10 minutes after the extraction, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand, centrifuge it at 4000 r / min for 10 minutes, dry the precipitate, and obtain crude protein from sturgeon bladder;

[0038] S3, extraction of crude protein liquid from sturgeon meat: wash the sturgeon meat, remove the surface connective tissue and fat, chop into small pieces, wash with distilled water 2-3 times to remove blood and impurities, then add to a wall breaking machine to make minced meat, add 100g minced meat to 700g distilled water, then add 2.5g cysteine ​​and 1.5g pepsin, and perform enzymolysis at a stirring rate of 100r / min and a temperature of 33°C for 5h. After the enzymolysis is completed, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, collect the supernatant, and obtain the crude protein liquid from sturgeon meat;

[0039] S4, mixed enzymolysis: 130g of the crude protein liquid of sturgeon skin in step S1, 25g of the crude protein of sturgeon maw in step S2, and 100g of the crude protein liquid of sturgeon meat in step S3 were mixed evenly to obtain a mixed solution, and then 0.765g of glutathione, 0.765g of neutral protease, and 3.315g of papain were added, and mixed enzymolysis was performed at 48°C for 1.5h. After the enzymolysis was completed, the mixture was centrifuged at 4000r / min for 10min, and the supernatant was taken to obtain an enzymolysis product;

[0040] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 minutes 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 a highly effective antioxidant sturgeon protein peptide.

[0041] Example 2

[0042] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

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

[0044] S2. Extraction of crude protein from sturgeon bladder: clean the sturgeon bladder and chop it into small pieces of 5 mm×5 mm, then immerse it in a sodium hydroxide solution with a concentration of 0.1 mol / L at a temperature of 4°C for 24 hours, wash it with distilled water until it is neutral, then add 100 g of sturgeon bladder to 1 L of acetic acid solution with a concentration of 0.5 mol / L, stir and extract it at 4°C for 42 hours, centrifuge it at 4000 r / min for 10 minutes after the extraction, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand and centrifuge it at 4000 r / min for 10 minutes, dry the precipitate to obtain crude protein from sturgeon bladder;

[0045] S3, extraction of crude protein liquid from sturgeon meat: wash the sturgeon meat, remove the surface connective tissue and fat, chop into small pieces, wash with distilled water 2-3 times to remove blood and impurities, then add to a wall breaking machine to make minced meat, add 100g minced meat to 500g distilled water, then add 2g cysteine ​​and 1g pepsin, and perform enzymolysis at a stirring rate of 80r / min and a temperature of 30°C for 6h. After the enzymolysis is completed, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, collect the supernatant, and obtain the crude protein liquid from sturgeon meat;

[0046] S4, mixed enzymolysis: 100g of the crude protein liquid of sturgeon skin in step S1, 20g of the crude protein of sturgeon maw in step S2, and 70g of the crude protein liquid of sturgeon meat in step S3 are mixed evenly to obtain a mixed solution, and then 0.38g of glutathione, 0.38g of neutral protease, and 1.9g of papain are added, and mixed enzymolysis is performed at 45°C for 2h. After the enzymolysis is completed, the mixture is centrifuged at 4000r / min for 10min, and the supernatant is taken to obtain an enzymolysis product;

[0047] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 minutes 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 a highly effective antioxidant sturgeon protein peptide.

[0048] Example 3

[0049] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

[0050] S1. Extraction of crude protein liquid from sturgeon skin: wash the sturgeon skin and chop it into small pieces of 5 mm × 5 mm, then soak it in a sodium chloride solution with a mass concentration of 6%, the soaking temperature is 10° C., the time is 12 h, wash it with distilled water after soaking, then add it to petroleum ether, extract it at 8° C. for 24 h, and obtain defatted sturgeon skin after extraction; add 100 g of defatted sturgeon skin to 1.5 L of 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, enzymolyze it at 35° C. for 2 h, and after the enzymolysis is completed, deactivate the enzyme in boiling water for 10 min, filter, centrifuge the filtrate at 4000 r / min for 10 min, take the supernatant, and obtain crude protein liquid from sturgeon skin;

[0051] S2. Extraction of crude protein from sturgeon bladder: clean the sturgeon bladder and chop 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 hours, wash it with distilled water until it is neutral, then add 100 g of sturgeon bladder to 1.5 L of acetic acid solution with a concentration of 0.8 mol / L, stir and extract it at 6°C for 36 hours, centrifuge it at 4000 r / min for 10 minutes after the extraction, collect the supernatant, add sodium chloride to a concentration of 1.2 mol / L for salting out, let it stand and centrifuge it at 4000 r / min for 10 minutes, dry the precipitate to obtain crude protein from sturgeon bladder;

[0052] S3, extraction of crude protein liquid from sturgeon meat: wash the sturgeon meat, remove the surface connective tissue and fat, chop into small pieces, wash with distilled water 2-3 times to remove blood and impurities, then add to a wall breaking machine to make minced meat, add 100g minced meat to 800g distilled water, then add 3g cysteine ​​and 2g pepsin, and perform enzymolysis at a stirring rate of 120r / min and a temperature of 35°C for 4h. After the enzymolysis is completed, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, collect the supernatant, and obtain the crude protein liquid from sturgeon meat;

[0053] S4, mixed enzymolysis: 150g of the crude protein liquid of sturgeon skin in step S1, 30g of the crude protein of sturgeon maw in step S2, and 120g of the crude protein liquid of sturgeon meat in step S3 are mixed evenly to obtain a mixed solution, and then 1.2g of glutathione, 1.2g of neutral protease, and 4.5g of papain are added, and mixed enzymolysis is performed at 50°C for 1h. After the enzymolysis is completed, the mixture is centrifuged at 4000r / min for 10min, and the supernatant is taken to obtain an enzymolysis product;

[0054] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 15 minutes 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 a highly effective antioxidant sturgeon protein peptide.

[0055] Comparative Example 1

[0056] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

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

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

[0059] S3, mixed enzymolysis: 25g of sturgeon maw crude protein in step S1 and 100g of sturgeon meat crude protein in step S2 are mixed evenly to obtain a mixed solution, and then 0.375g of glutathione, 0.375g of neutral protease, and 1.625g of papain are added, and mixed enzymolysis is performed at 48°C for 1.5h. After the enzymolysis is completed, the mixture is centrifuged at 4000r / min for 10min, and the supernatant is taken to obtain an enzymolysis product;

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

[0061] Compared with Example 1, this comparative example did not add sturgeon skin crude protein liquid.

[0062] Comparative Example 2

[0063] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

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

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

[0066] S3, mixed enzymolysis: 130g of the crude protein solution of sturgeon skin in step S1 and 25g of the crude protein of sturgeon maw in step S2 were mixed evenly to obtain a mixed solution, and then 0.465g of glutathione, 0.465g of neutral protease, and 2.015g of papain were added, and mixed enzymolysis was performed at 48°C for 1.5h. After the enzymolysis was completed, the mixture was centrifuged at 4000r / min for 10min, and the supernatant was taken to obtain an enzymolysis product;

[0067] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 minutes 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 a highly effective antioxidant sturgeon protein peptide.

[0068] Compared with Example 1, no sturgeon meat crude protein liquid was added in this comparative example.

[0069] Comparative Example 3

[0070] A method for preparing a highly effective antioxidant sturgeon protein peptide comprises the following steps:

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

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

[0073] S3, extraction of crude protein liquid from sturgeon meat: wash the sturgeon meat, remove the surface connective tissue and fat, chop into small pieces, wash with distilled water 2-3 times to remove blood and impurities, then add to a wall breaking machine to make minced meat, add 100g minced meat to 700g distilled water, then add 1.5g pepsin, and perform enzymolysis at a stirring rate of 100r / min and a temperature of 33°C for 5h. After the enzymolysis is completed, inactivate the enzyme in boiling water for 10min, centrifuge at 4000r / min for 10min, collect the supernatant, and obtain the crude protein liquid from sturgeon meat;

[0074] S4, mixed enzymolysis: 130g of the crude protein liquid of sturgeon skin in step S1, 25g of the crude protein of sturgeon maw in step S2, and 100g of the crude protein liquid of sturgeon meat in step S3 were mixed evenly to obtain a mixed solution, and then 0.765g of neutral protease and 3.315g of papain were added, and mixed enzymolysis was performed at 48°C for 1.5h. After the enzymolysis was completed, the mixture was centrifuged at 4000r / min for 10min, and the supernatant was taken to obtain an enzymolysis product;

[0075] S5. Inactivate the enzyme in the obtained enzymatic hydrolysis product in a boiling water bath for 10 minutes 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 a highly effective antioxidant sturgeon protein peptide.

[0076] Compared with Example 1, in this comparative example, tea polyphenols were not added when preparing the crude protein liquid of sturgeon skin, cysteine ​​was not added when preparing the crude protein liquid of sturgeon meat, 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 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; 1 mL of 40 μmol / L DPPH free radical ethanol solution was added to 4 mL of distilled water and the above sturgeon protein peptide solution samples, respectively, and the mixture was allowed to stand at 25° C. for 30 min. The absorption values ​​of A blank and A sample were measured at 517 nm, respectively. The calculation formula for scavenging DPPH free radicals was:

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

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

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

[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 a highly effective 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. The obtained enzymatic hydrolysis product is first subjected to enzyme inactivation treatment to obtain an enzymatic hydrolysis solution, and then the enzymatic hydrolysis solution is subjected to ultrafiltration and vacuum drying in sequence to obtain a highly effective antioxidant sturgeon protein peptide.

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: In step S4, the mass ratio of the sturgeon skin crude protein liquid, the sturgeon swim bladder crude protein, and the sturgeon meat crude protein liquid is 100-150:20-30:70-120.

8. The preparation method according to claim 1, characterized in that: In step S4, 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, the temperature of the mixed enzymolysis is 45-50° C., and the time is 1-2h.

9. 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.

10. A highly effective antioxidant sturgeon protein peptide prepared by the method according to any one of claims 1 to 9.

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