Sturgeon extract rich in antioxidant peptides, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a sturgeon extract rich in antioxidant peptides, and a preparation method therefor and the use thereof. The sturgeon extract of the present invention comprises an amino acid, polypeptide and protein from sturgeon meat and / or roe. The total amount of the amino acid, polypeptide and protein is 20% or more based on the dry matter content. On the basis that the total mass of the amino acid, polypeptide and protein is 100%, the molecular weight distribution of the amino acid, polypeptide and protein is as follows: the content of the component with a molecular weight less than 500 Da is 9%-60%; the content of the component with a molecular weight of 500 Da-1000 Da is 10%-30%; the content of the component with a molecular weight of 1000 Da-5000 Da is 5%-60%; and the content of the component with a molecular weight greater than 5000 Da is 5%-52%. The sturgeon extract of the present invention is rich in a variety of antioxidant peptides, has a relatively high antioxidant activity and also has the functions of protecting the liver and enhancing immunity.

Description

Sturgeon extract rich in antioxidant peptides, preparation method and application thereof Technical Field

[0001] The present invention relates to a preparation method and application of a natural active substance, specifically to a sturgeon extract rich in antioxidant peptides, a preparation method and application thereof, and belongs to the field of extraction technology of natural active substances and their application in medicine and health products. Background Art

[0002] Sturgeons are among the earliest living vertebrates, and their genomes carry unique proteomic information, resulting in sturgeon tissues and roe rich in active ingredients such as amino acids, active peptides, and antioxidant enzymes. Caviar, a key derivative of sturgeon, boasts both high nutritional value and significant economic value, making it highly sought after by consumers. China, a major participant in the global sturgeon trade, boasts abundant sturgeon aquaculture resources, but limited processing methods and a shortage of downstream derivatives have hindered the core competitiveness of these products. Because caviar provides a variety of high-value nutrients essential for fish development, caviar is nutritiously rich, including various amino acids, active proteins, highly unsaturated fatty acids (EPA and DHA), inorganic salts, vitamins, and trace elements such as calcium, copper, iron, and selenium. The combined protein and lipid content of sturgeon roe exceeds that of other fish eggs. To prevent the oxidative breakdown of easily oxidized substances such as highly unsaturated fatty acids, sturgeon tissues contain a significant endogenous antioxidant system, which maintains redox homeostasis.

[0003] Under normal circumstances, reactive oxygen species (ROS) are byproducts of aerobic metabolic cycles and play an important role in immune responses, signal transduction, mitosis, and other processes. However, when the body is exposed to specific environments or toxic substances, excessive ROS concentrations can cause "oxidative stress" hazards to biological macromolecules such as lipids, DNA, and proteins, disrupting the normal structure and function of cells. The antioxidant system of organisms includes enzymes and non-enzymatic small molecules. Fish meat, fish eggs, and their hydrolysates have been shown to be rich in a variety of natural biological antioxidants such as amino acids, peptides, and active proteins. In addition, various intrinsic factors such as fish species, age, feeding behavior, and environmental factors can affect their antioxidant system.

[0004] However, the composition of caviar is complex, and hydrophobic interactions between biomacromolecules within confined spaces cause different components to intermingle. For example, yolk yolk is primarily located in the lipid-rich egg yolk, naturally existing as a low-solubility yolk lipoprotein-yolk high phospholipid protein complex. This poses a significant challenge to separating various components with specific biological functions. Therefore, there is an urgent need to develop efficient extraction methods and processes for key active ingredients in sturgeon roe and fish meat, providing reliable, stable, and functional key active ingredients for the development of sturgeon roe derivatives.

[0005] There are two methods and approaches for preparing bioactive peptides, such as antioxidant peptides: one is to extract various naturally active peptide substances inherent in organisms; the other is to obtain bioactive peptides with various physiological functions through enzymatic hydrolysis. However, enzymatic hydrolysis is a complex process that may produce some unidentified products or be uncontrollable, making subsequent separation and purification more difficult and reducing the biosafety of the final product. Furthermore, enzymatic hydrolysis is often limited by the enzyme's optimal hydrolysis temperature and pH, resulting in dependence on multiple equipment and high production costs. Direct extraction methods use various separation and purification techniques to extract active peptides from organisms. Active peptides prepared using direct extraction methods are safe, reliable, cost-effective, and efficient because they avoid destroying the inherent active components of the organism.

[0006] CN103923963A discloses a method for preparing fish skin collagen and ACE inhibitory peptides. The process involves washing the fish skin and freezing and crushing it. The fish skin powder is enzymatically hydrolyzed and inactivated with metallopeptidase Protease A. The enzymatic hydrolyzate is filtered to obtain a supernatant and a filter residue. The supernatant is concentrated and dried to obtain fish skin collagen. The obtained collagen is then enzymatically hydrolyzed and inactivated with AS1398 enzyme. The supernatant is cooled and centrifuged to obtain the ACE inhibitory peptide after concentration under reduced pressure and freeze-drying. This method uses two enzymes in combination. The enzymatic hydrolysis time of Protease A is 3-7 hours, and the enzymatic hydrolysis temperature of AS1398 is 40-55°C. Therefore, the process relies on temperature control equipment and has a long preparation time. The preparation environment requirements are relatively high. The use of enzymes also greatly increases the cost, which also limits its large-scale application in industry.

[0007] CN101240014B discloses a method for preparing a physiologically active lipopeptide complex. This patent involves crushing or pulverizing tissue from a member of the family Muricidae, order Neogastropoda, phylum Mollusca, and adjusting the pH to 4.0-6.5 with hydrochloric acid or an organic acid. The extracts are combined and cooled to a temperature below the extraction temperature to precipitate. The supernatant is collected, concentrated to remove the solvent, purified with petroleum ether, and then separated and eluted using a dextran gel chromatography column to obtain the lipopeptide complex. The prepared lipopeptide complex has the function of preventing, improving, and treating hypertension. However, this method is complex in preparation and uses reagents such as hydrochloric acid and petroleum ether, which poses certain biosafety risks. The high cost further limits its large-scale industrial application.

[0008] CN109430883A discloses a method for preparing a free peptide extract from marine fish. The patent involves mincing the pre-treated marine fish, then adding water for at least one ultrasonic extraction, collecting the extract, concentrating it, and drying it. The free peptide extract from marine fish prepared by this preparation method has high levels of histidine dipeptide, glutathione, and taurine, and can effectively improve chloasma. However, there is no quantitative test data on the extract's antioxidant and melanin-clearing abilities, and its practicality remains to be verified.

[0009] At present, there is no research in the existing technology on the process of directly using the crushing extraction method to prepare sturgeon meat and fish egg extracts rich in antioxidant peptides. In addition, the current enzymatic hydrolysis preparation process is complicated, the extraction time is long, the equipment is highly dependent, and the cost is high, which limits its large-scale industrial production. Summary of the Invention

[0010] To solve the above technical problems, the present invention aims to provide a sturgeon extract and its preparation method and application. Another object of the present invention is to provide a polypeptide composition and its application.

[0011] In one aspect, the present invention provides a sturgeon extract comprising amino acids, polypeptides, and proteins from sturgeon meat and / or roe; the total amount of amino acids, polypeptides, and proteins, calculated on a dry matter basis, is greater than 20% (typically 20-90%, and may be further purified to increase the content);

[0012] Among them, taking the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 9-60%; the molecular weight is 500Da-1000Da, and its content is 10-30%; the molecular weight is 1000Da-5000Da, and its content is 5-60%; the molecular weight is greater than 5000Da, and its content is 5-52%.

[0013] The sturgeon extract preferably contains at least 40% amino acids, polypeptides, and proteins, for example, 40-90% of the total amount, calculated on a dry matter basis. In addition to amino acids, polypeptides, and proteins, the sturgeon extract also contains fats, minerals, vitamins, unsaturated fatty acids, and the like. The sturgeon extract can be further purified by defatting or other steps to increase the amino acid, polypeptide, and protein content.

[0014] Among the above-mentioned sturgeon extracts, preferably, it is a sturgeon meat extract, wherein, based on the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and the content thereof is 11-60%; the molecular weight is 500Da-1000Da, and the content thereof is 15-30%; the molecular weight is 1000Da-5000Da, and the content thereof is 5-60%; the molecular weight is greater than 5000Da, and the content thereof is 10-52%.

[0015] Preferably, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 20-60%; the molecular weight is 500Da-1000Da, and its content is 20-30%; the molecular weight is 1000Da-5000Da, and its content is 5-50%; the molecular weight is greater than 5000Da, and its content is 10-20%.

[0016] Among the above-mentioned sturgeon extracts, preferably, it is a sturgeon egg extract, wherein, based on the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: those with a molecular weight of less than 500Da, the content of which is 15-40%; those with a molecular weight of 500Da-1000Da, the content of which is 10-30%; those with a molecular weight of 1000Da-5000Da, the content of which is 20-55%; and those with a molecular weight of greater than 5000Da, the content of which is 5-50%.

[0017] Preferably, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 15-40%; the molecular weight is 500Da-1000Da, and its content is 11-30%; the molecular weight is 1000Da-5000Da, and its content is 20-55%; the molecular weight is greater than 5000Da, and its content is 5-35%.

[0018] According to a specific embodiment of the present invention, preferably, the detection process of the sturgeon extract is as shown in the flow chart of Figure 1: first, the sample is purified and reductively alkylated, and then the sample is desalted. Thereafter, the treated sample is analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain a raw file of the original mass spectrometry result, and then the peptide sequence analysis result is obtained through de novo analysis.

[0019] The sturgeon extract preferably contains one or more polypeptides shown in any one of SEQ ID NOs: 1-30.

[0020] In the above-mentioned sturgeon extract, preferably, when its concentration is 0.2-50 mg / mL, the scavenging rate of hydroxyl radicals is 0.8%-97.3%;

[0021] Preferably, when the concentration of the extract is 10-50 mg / mL, the scavenging rate of hydroxyl radicals is 1.8%-97.3%.

[0022] In another aspect, the present invention provides a method for preparing the sturgeon extract, wherein the preparation method comprises the following steps:

[0023] Adding fresh sturgeon meat and / or fish eggs to water, breaking the fish wall or homogenizing to obtain a homogenate;

[0024] The homogenate is centrifuged or plate-and-frame filtered to remove the residue and the extract is collected;

[0025] The extract is then filtered through a membrane to obtain a liquid sturgeon extract; or further dried to obtain a solid sturgeon extract.

[0026] In the above preparation method, preferably, the mass ratio of the fish meat and / or fish eggs to water is 1:(0.5-100), preferably 1:(1-30);

[0027] The preparation temperature of the homogenate is 4-50°C;

[0028] Preferably, after cell wall breaking or homogenization, further ultrasonication may be performed to obtain a homogenate;

[0029] Preferably, the cell wall breaking process is performed for 3-30 minutes and 3-20 times.

[0030] Preferably, the homogenization is carried out at a temperature of 4-30°C, a pressure of 5-60 MPa, and a homogenization frequency of 1-10 times;

[0031] Preferably, the ultrasonic treatment is carried out at an ultrasonic frequency of 5-50 kHz, an ultrasonic power of 50-1000 W, and an ultrasonic time of 3-60 min;

[0032] The bag or plate-and-frame filtration conditions used in the centrifugation are 400-600 mesh, and the centrifugation time is 20-30 min;

[0033] The filter membrane specification used in the membrane filtration is 0.05 μm-5 μm, preferably 0.05-3 μm;

[0034] The drying method includes one or a combination of two or more of natural drying, spray drying or freeze drying.

[0035] Compared with the existing technology, the preparation method of sturgeon extract provided by the present invention is simple and efficient, does not require additional temperature control equipment, and is more conducive to large-scale production. In addition, the sturgeon extract obtained according to the preparation method of the present invention has better free radical scavenging ability, so it can be applied to various antioxidant scenarios, such as effectively playing the role of wrinkle removal and firming in the aging population due to free radical oxidative damage, and has the effect of protecting the liver and enhancing immunity in the population with liver damage induced by chronic inflammation and immunosuppression.

[0036] The sturgeon extract obtained by the preparation method of the present invention exhibits excellent antioxidant, liver protection and immune enhancement functions, has high application value, and provides a material basis for the preparation of new medicines, health products and cosmetics.

[0037] On the other hand, the present invention provides a use of the above-mentioned sturgeon extract, or the sturgeon extract prepared by the above-mentioned preparation method, in the preparation of products for antioxidant, anti-inflammatory, liver protection and / or immune enhancement. Preferably, the product is a medicine, health product or cosmetic.

[0038] In another aspect, the present invention provides a polypeptide composition comprising a combination of one or more of the polypeptides shown in any one of SEQ ID NOs: 1-30.

[0039] On the other hand, the present invention provides a use of the above-mentioned polypeptide composition in the preparation of antioxidant, anti-inflammatory, liver protection and / or immune enhancement products. Preferably, the product is a medicine, health product or cosmetic.

[0040] In another aspect, the present invention provides a medicine, a health product or a cosmetic, wherein the medicine, the health product or the cosmetic comprises the sturgeon extract and / or the polypeptide composition.

[0041] Medicine and health products of the present invention further include pharmaceutically acceptable excipients or carriers, including but not limited to solvents, suspending agents, buffers, materials that make the preparation isotonic with blood, stabilizers or antimicrobial preservatives. The interpolation of excipients should remain at a minimum. When using excipients, they should not adversely affect the stability, bioavailability, safety or efficacy of polymers and / or activating agents, or cause toxicity or excessive local irritation. There should not be any incompatibility between any components of the dosage form.

[0042] The health care products described in the present invention may refer to products used for health care and health preservation in the general sense.

[0043] The medicine or health care product of the present invention can be prepared in the following preparation forms, for example, oral preparations (such as granules, powders, capsules, pills, tablets, dry syrups, liquid preparations), or medicines or health care products prepared by mixing with other known additives, such as: carriers, disintegrants, sustained-release agents, excipients, fillers, coating agents, binders, lubricants, antioxidants, coating agents, colorants, flavorings, sweeteners, sucrose substitutes, nutritional enhancers, flavorings, surfactants, plasticizers, pH regulators, fresheners, suspending agents, defoaming agents, preservatives, thickeners, solubilizing agents, etc., which are prepared according to conventional methods or added to medicines or health care products as functional components or additives.

[0044] The cosmetic products of the present invention are designed to be applied to the body in an appropriate amount ("cosmetically effective amount") to improve the appearance or beautify, maintain, condition, cleanse, color or protect the skin, nails or hair.

[0045] The cosmetics of the present invention include pharmaceutically or cosmetically acceptable excipients or carriers, which may include but are not limited to repairing skin barrier function agents, moisturizers, emollients, emulsifiers, thickeners, moisturizers, pH regulators, antioxidants, preservatives, vehicles, or mixtures thereof. The excipients or carriers used have affinity for the skin, are well tolerated, stable, and are used in amounts sufficient to provide the desired consistency and ease of application. In addition, the cosmetics of the present invention may contain other ingredients, such as fragrances, colorants, and other components known in the prior art for use in cosmetics. The cosmetics of the present invention can be formulated in several forms, including but not limited to solutions, aerosols, and non-aerosol sprays, creams, powders, mousses, lotions, gels, sticks, ointments, pastes, shampoos, shower gels, bath preparations, or facial cleansers and lotions.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] 1. The sturgeon extract provided by the present invention has excellent antioxidant activity and can be used in wrinkle-removing and firming cosmetics. In addition, it also has the functions of protecting the liver and enhancing immunity.

[0048] 2. The preparation method of the sturgeon extract provided by the present invention is simple and efficient, does not require temperature control equipment, etc., has low equipment requirements, and reduces production energy consumption.

[0049] 3. The preparation method of the sturgeon extract provided by the present invention does not require any hydrolase, has low production cost, and can be operated continuously online. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a schematic diagram of the peptide sequence detection process of sturgeon extract.

[0051] FIG2 shows the hydroxyl radical scavenging rate of the sturgeon extract prepared in Example 1 and the specific polypeptides therein.

[0052] FIG3 shows the polypeptide molecular weight distribution of the sturgeon extract powder obtained by the direct crushing method in Example 1 and the extract powder obtained by enzymatic hydrolysis for 2 h in Comparative Example 2.

[0053] FIG4 shows the hydroxyl radical scavenging rates of the sturgeon extracts prepared in Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0054] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0055] The features and exemplary embodiments of various aspects of the present invention and comparative examples are described in detail below. In the detailed description below, many specific details are set forth in order to provide a comprehensive understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be practiced without some of these specific details. The following description of the embodiments is intended only to provide a better understanding of the present invention by illustrating examples of the present invention.

[0056] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0057] Example 1

[0058] The preparation of sturgeon extract in this embodiment includes the following steps:

[0059] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0060] (2) Place 2 kg of fresh and clean sturgeon meat or fish eggs in a wall breaking machine, add 20 kg of deionized water, and break the wall five times in succession, with a single breaking time of 5 min and an interval of 30 s to obtain a uniform and fine homogenate;

[0061] (3) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0062] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0063] (5) Subsequently, spray drying is performed to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, polypeptides, and proteins from sturgeon meat or fish eggs; the total amount of amino acids, polypeptides, and proteins in the obtained sturgeon meat extract is 70% based on dry matter content.

[0064] In the obtained sturgeon roe extract, the total amount of amino acids, polypeptides and proteins is 80% based on dry matter content.

[0065] (6) Performing a polypeptide molecular weight distribution test on the sturgeon extract powder obtained in step (5).

[0066] (7) Sturgeon meat extract, fish egg extract, and identified specific antioxidant peptide powder (sequence information is shown in Table 1) were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested. The results are shown in Figure 2. As can be seen from Figure 2, the antioxidant capacity of different specific peptides varies greatly, and sturgeon meat extract has the highest hydroxyl radical scavenging capacity.

[0067] The test method refers to the hydroxyl radical scavenging ability test kit (Solabo, product number BC1320): first preheat the microplate reader for more than 30 minutes, adjust the wavelength to 536nm, and zero with distilled water. Prepare a 10mg / mL vitamin C solution as a positive control group. Add 10uL of sturgeon extract (assay group), vitamin C (control group) or H2O (blank group) to 190uL of hydroxyl radical solution, vortex to mix, place in a 37°C constant temperature incubator for accurate reaction for 60 minutes, and measure the absorbance at 536nm. The hydroxyl radical scavenging rate of the sample is calculated as follows:

[0068] Hydroxyl radical scavenging rate D% = (A 测试 -A 对照 ) / (A 空白 -A 对照 ) x 100%. The molecular weight distribution of the sturgeon meat extract is as follows: peptides with a molecular weight of less than 500Da account for 39.2%; peptides with a molecular weight of 500Da-1000Da account for 18.0%; peptides with a molecular weight of 1000Da-5000Da account for 12.9%; and peptides with a molecular weight of greater than 5000Da account for 29.9%. The hydroxyl radical scavenging rate is 18.88%.

[0069] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 20.3%; the molecular weight is 500Da-1000Da, and its content is 10.2%; the molecular weight is 1000Da-5000Da, and its content is 27.5%; the molecular weight is greater than 5000Da, and its content is 42.0%. The scavenging rate of hydroxyl free radicals is 2.0%.

[0070] Table 1 Specific polypeptide sequences

[0071] Example 2

[0072] The sturgeon extract prepared in this embodiment includes the following steps:

[0073] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0074] (2) 2 kg of fresh and clean sturgeon meat or fish eggs were placed in a wall breaking machine, 20 kg of deionized water was added, and the wall was broken three times in succession, with a single wall breaking time of 5 min and an interval of 30 s. The temperature was controlled at 25 ° C to obtain a homogenate. The homogenate was then ultrasonicated at an ultrasonic frequency of 25 kHz, an ultrasonic power of 450 W, and an ultrasonic time of 30 min to obtain a uniform and fine homogenate.

[0075] (3) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0076] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0077] (5) subsequently spray-drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0078] The obtained sturgeon meat extract has a total content of amino acids, polypeptides and proteins of 78% based on dry matter content.

[0079] In the obtained sturgeon roe extract, the total amount of amino acids, polypeptides and proteins is 85% based on dry matter content.

[0080] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0081] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0082] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the molecular weight is less than 500Da, and its content is 50.2%; the molecular weight is 500Da-1000Da, and its content is 25.7%; the molecular weight is 1000Da-5000Da, and its content is 5.9%; the molecular weight is greater than 5000Da, and its content is 18.2%; the scavenging rate of hydroxyl free radicals is 25.44%.

[0083] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 25.4%; the molecular weight is 500Da-1000Da, and its content is 12.8%; the molecular weight is 1000Da-5000Da, and its content is 20.5%; the molecular weight is greater than 5000Da, and its content is 41.3%. The scavenging rate of hydroxyl free radicals is 3.6%.

[0084] Example 3

[0085] The sturgeon extract prepared in this embodiment includes the following steps:

[0086] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0087] (2) 2 kg of fresh and clean sturgeon meat or fish eggs were placed in a wall breaking machine, 2 kg of deionized water was added, and the wall was broken three times in succession, with a single wall breaking time of 5 min and an interval of 30 s. The temperature was controlled at 25 ° C to obtain a homogenate. The homogenate was then ultrasonicated at an ultrasonic frequency of 25 kHz, an ultrasonic power of 450 W, and an ultrasonic time of 30 min to obtain a uniform and fine homogenate.

[0088] (3) Centrifuge the homogenate for 20 min using a 500-mesh filter bag, remove the residue, and collect the extract;

[0089] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0090] (5) subsequently spray-drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0091] The obtained sturgeon meat extract has a total content of amino acids, polypeptides and proteins of 48% based on dry matter content.

[0092] In the obtained sturgeon roe extract, the total amount of amino acids, polypeptides and proteins is 62% based on dry matter content.

[0093] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0094] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0095] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the molecular weight is less than 500Da, and its content is 9.6%; the molecular weight is 500Da-1000Da, and its content is 18.4%; the molecular weight is 1000Da-5000Da, and its content is 43.8%; the molecular weight is greater than 5000Da, and its content is 28.2%; the scavenging rate of hydroxyl free radicals is 10.5%.

[0096] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 15.7%; the molecular weight is 500Da-1000Da, and its content is 24.5%; the molecular weight is 1000Da-5000Da, and its content is 50.8%; the molecular weight is greater than 5000Da, and its content is 9%. The scavenging rate of hydroxyl free radicals is 8.6%.

[0097] Example 4

[0098] The sturgeon extract prepared in this embodiment includes the following steps:

[0099] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0100] (2) 2 kg of fresh and clean sturgeon meat or fish eggs were placed in a wall breaking machine, 20 kg of deionized water was added, and the wall was broken three times in succession, with a single wall breaking time of 5 min and an interval of 30 s. The temperature was controlled at 25°C to obtain a homogenate. The homogenate was then ultrasonicated at an ultrasonic frequency of 15 kHz, an ultrasonic power of 400 W, and an ultrasonic time of 20 min to obtain a uniform and fine homogenate.

[0101] (3) Centrifuge the homogenate for 20 min using a 500-mesh filter bag, remove the residue, and collect the extract;

[0102] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0103] (5) subsequently spray-drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0104] In the obtained sturgeon meat extract, the total amount of amino acids, polypeptides and proteins is 50% based on dry matter content.

[0105] In the obtained sturgeon roe extract, the total amount of amino acids, polypeptides and proteins is 45% based on dry matter content.

[0106] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0107] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0108] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the molecular weight is less than 500Da, and its content is 24.6%; the molecular weight is 500Da-1000Da, and its content is 22.9%; the molecular weight is 1000Da-5000Da, and its content is 31.7%; the molecular weight is greater than 5000Da, and its content is 20.8%; the scavenging rate of hydroxyl free radicals is 15.6%.

[0109] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 15.4%; the molecular weight is 500Da-1000Da, and its content is 12.7%; the molecular weight is 1000Da-5000Da, and its content is 22.6%; the molecular weight is greater than 5000Da, and its content is 49.3%. The scavenging rate of hydroxyl free radicals is 3.8%.

[0110] Example 5

[0111] The sturgeon extract prepared in this embodiment includes the following steps:

[0112] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0113] (2) 2 kg of fresh and clean sturgeon meat or fish eggs were placed in a wall breaking machine, 2 kg of deionized water was added, and the wall was broken three times in succession. The single wall breaking time was 5 min, the interval was 30 s, and the temperature was controlled at 25 °C to obtain a homogenate. The homogenate was then subjected to high-pressure homogenization. The parameters were set as follows: temperature 25 °C, pressure 40 MPa, homogenization times 5 times, and interval 5 s;

[0114] (3) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0115] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0116] (5) subsequently spray-drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0117] In the obtained sturgeon meat extract, the total amount of amino acids, polypeptides and proteins is 84% ​​based on dry matter content.

[0118] The obtained sturgeon roe extract has a total content of amino acids, polypeptides and proteins of 72% based on dry matter content.

[0119] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0120] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0121] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the molecular weight is less than 500Da, and its content is 24.7%; the molecular weight is 500Da-1000Da, and its content is 20.0%; the molecular weight is 1000Da-5000Da, and its content is 40.5%; the molecular weight is greater than 5000Da, and its content is 14.8%; the scavenging rate of hydroxyl free radicals is 23.05%.

[0122] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 34.8%; the molecular weight is 500Da-1000Da, and its content is 11.5%; the molecular weight is 1000Da-5000Da, and its content is 20.4%; the molecular weight is greater than 5000Da, and its content is 33.3%. The scavenging rate of hydroxyl free radicals is 10.5%.

[0123] Example 6

[0124] The sturgeon extract prepared in this embodiment includes the following steps:

[0125] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0126] (2) 2 kg of fresh and clean sturgeon meat or fish eggs were placed in a wall breaking machine, 50 kg of deionized water was added, and the wall was broken three times in succession. The single wall breaking time was 5 min, the interval was 30 s, and the temperature was controlled at 25 °C to obtain a homogenate. The homogenate was then subjected to high-pressure homogenization. The parameters were set as follows: temperature 25 °C, pressure 40 MPa, homogenization times 3 times, and intervals of 5 s to obtain a uniform and fine homogenate.

[0127] (3) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0128] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0129] (5) subsequently spray-drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract powder includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0130] In the obtained sturgeon meat extract, the total amount of amino acids, polypeptides and proteins is 69% based on dry matter content.

[0131] The obtained sturgeon roe extract has a total content of amino acids, polypeptides and proteins of 71% based on dry matter content.

[0132] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0133] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0134] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the content of polypeptides with a molecular weight of less than 500Da is 12.4%; the content of polypeptides with a molecular weight of 500Da-1000Da is 20.8%; the content of polypeptides with a molecular weight of 1000Da-5000Da is 51.1%; the content of polypeptides with a molecular weight of greater than 5000Da is 59.6%; and the scavenging rate for hydroxyl free radicals is 1.8%.

[0135] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 23%; the molecular weight is 500Da-1000Da, and its content is 11.4%; the molecular weight is 1000Da-5000Da, and its content is 36.8%; the molecular weight is greater than 5000Da, and its content is 28.8%. The scavenging rate of hydroxyl free radicals is 5.4%.

[0136] Comparative Example 1

[0137] The sturgeon extract prepared in this comparative example comprises the following steps:

[0138] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0139] (2) 2 kg of fresh and clean sturgeon meat or roe were added to 4 kg of deionized water and ultrasonicated at a frequency of 15 kHz, a power of 400 W, and a duration of 5 min, but no uniform and fine homogenate was obtained;

[0140] (3) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0141] (4) filtering the extract through a membrane with a pore size of 3 μm to obtain a sturgeon extract stock solution;

[0142] (5) subsequently spray-drying to obtain a sturgeon extract powder comprising amino acids, polypeptides, and proteins derived from sturgeon meat or roe;

[0143] The obtained sturgeon meat extract has a total content of amino acids, polypeptides and proteins of 47% based on dry matter content.

[0144] The obtained sturgeon roe extract has a total content of amino acids, polypeptides and proteins of 58% based on dry matter content.

[0145] (6) performing a polypeptide molecular weight distribution test on the extract powder in step (5);

[0146] (7) Sturgeon meat extract and fish egg extract powder were dissolved in H2O to 10 mg / mL, and the hydroxyl radical scavenging rate was tested;

[0147] The molecular weight distribution of polypeptides in the sturgeon meat extract is as follows: the content of polypeptides with a molecular weight of less than 500Da is 5.8%; the content of polypeptides with a molecular weight of 500Da-1000Da is 10.5%; the content of polypeptides with a molecular weight of 1000Da-5000Da is 25.4%; the content of polypeptides with a molecular weight of greater than 5000Da is 58.3%; and the scavenging rate of polypeptides for hydroxyl free radicals is 0.4%.

[0148] The molecular weight distribution of polypeptides in sturgeon egg extract is as follows: the molecular weight is less than 500Da, and its content is 10.5%; the molecular weight is 500Da-1000Da, and its content is 9.4%; the molecular weight is 1000Da-5000Da, and its content is 24.1%; the molecular weight is greater than 5000Da, and its content is 56%. The scavenging rate of hydroxyl free radicals is 1.2%.

[0149] Comparative Example 2

[0150] The sturgeon extract prepared in this comparative example comprises the following steps:

[0151] (1) Bleeding and cleaning the freshly caught sturgeon, removing the head, viscera, gills, and scales, and obtaining the sturgeon meat and roe, which were then washed with deionized water and set aside;

[0152] (2) Place 2 kg of fresh and clean sturgeon meat or fish eggs in a wall breaking machine, add 20 kg of deionized water, and break the wall five times in succession, with a single breaking time of 5 min and an interval of 30 s to obtain a uniform and fine homogenate;

[0153] (3) 1 wt% of proteases, including alcalase and trypsin, were added to the fish meat or fish egg homogenate and incubated at the enzyme's optimal working temperature for 1 or 2 h, respectively. The optimal working temperature for alcalase is 55°C, and the optimal working temperature for trypsin is 37°C. The mixture was then incubated at 90°C for 15 minutes to completely inactivate the enzymes.

[0154] (4) Centrifuge the homogenate for 20 min using a 400-mesh filter bag, remove the residue, and collect the extract;

[0155] (5) The extract was filtered through a membrane with a pore size of 3 μm to obtain the sturgeon extract solution.

[0156] (6) subsequently spray drying to obtain a sturgeon extract powder rich in antioxidant peptides, wherein the extract includes amino acids, peptides, and proteins from sturgeon meat or roe;

[0157] In the sturgeon meat extract obtained by enzymatic hydrolysis for 2 hours, the total amount of amino acids, polypeptides and proteins is 82% based on dry matter content.

[0158] In the sturgeon egg extract obtained by enzymatic hydrolysis for 2 hours, the total amount of amino acids, polypeptides and proteins is 74% based on dry matter content.

[0159] (7) Testing the molecular weight distribution of the extract powder in step (6)

[0160] (8) Conducting an in vitro antioxidant function test on the sturgeon extract solution in step (5).

[0161] The molecular weight distributions of peptides in the sturgeon extract powder obtained by the direct crushing method in Example 1 and the extract powder obtained by enzymatic hydrolysis for 2 hours in this comparative example are shown in Figure 3. For sturgeon meat extract, the direct crushing method yielded significantly higher levels of small peptides (MW < 0.5 kDa) than the enzymatic hydrolysis method. For fish egg extract, the small peptide contents of the extracts obtained by both methods were similar.

[0162] The hydroxyl radical scavenging efficiency of the sturgeon extract obtained by the direct crushing method in Example 1 of the present invention and the extract obtained by enzymatic hydrolysis in this comparative example were measured. The results are shown in Figure 4. The hydroxyl radical scavenging activity of the untreated fish meat and fish egg extracts in Example 1 is measured for the 0-hour enzymatic hydrolysis time. The hydroxyl radical scavenging efficiency of the extract obtained by enzymatic hydrolysis in this comparative example is measured for the 1- and 2-hour enzymatic hydrolysis times. As can be seen in Figure 4, the sturgeon extract obtained by the direct crushing method exhibits significantly greater hydroxyl radical scavenging efficiency than the enzymatic hydrolysis method.

[0163] The above embodiments and comparative examples show and describe the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only to illustrate the principles of the present invention, and is not to limit the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection required. Any person skilled in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should all be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A sturgeon extract comprising amino acids, polypeptides, and proteins derived from sturgeon meat and / or roe; the total amount of the amino acids, polypeptides, and proteins being greater than 20% based on dry matter content; in, Taking the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 9-60%; the molecular weight is 500Da-1000Da, and its content is 10-30%; the molecular weight is 1000Da-5000Da, and its content is 5-60%; the molecular weight is greater than 5000Da, and its content is 5-52%.

2. The sturgeon extract according to claim 1, which is a sturgeon meat extract, wherein Taking the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and the content thereof is 11-60%; the molecular weight is 500Da-1000Da, and the content thereof is 15-30%; the molecular weight is 1000Da-5000Da, and the content thereof is 5-60%; the molecular weight is greater than 5000Da, and the content thereof is 10-52%; Preferably, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 20-60%; the molecular weight is 500Da-1000Da, and its content is 20-30%; the molecular weight is 1000Da-5000Da, and its content is 5-50%; the molecular weight is greater than 5000Da, and its content is 10-20%.

3. The sturgeon extract according to claim 1, which is a sturgeon roe extract, wherein Taking the total mass of amino acids, polypeptides and proteins as 100%, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the content of amino acids with a molecular weight of less than 500Da is 15-40%; the content of amino acids with a molecular weight of 500Da-1000Da is 10-30%; the content of amino acids with a molecular weight of 1000Da-5000Da is 20-55%; the content of amino acids with a molecular weight of greater than 5000Da is 5-50%; Preferably, the molecular weight distribution of amino acids, polypeptides and proteins is as follows: the molecular weight is less than 500Da, and its content is 15-40%; the molecular weight is 500Da-1000Da, and its content is 11-30%; the molecular weight is 1000Da-5000Da, and its content is 20-55%; the molecular weight is greater than 5000Da, and its content is 5-35%.

4. The sturgeon extract according to claim 1, comprising one or more polypeptides represented by any one of SEQ ID NOs: 1-30.

5. The sturgeon extract according to claim 1, wherein the scavenging rate of the sturgeon extract against hydroxyl radicals is 0.8% to 97.3% at a concentration of 0.2 to 50 mg / mL; Preferably, when the concentration of the extract is 10-50 mg / mL, the scavenging rate of hydroxyl radicals is 1.8%-97.3%.

6. A method for preparing the sturgeon extract according to any one of claims 1 to 5, wherein: The preparation method comprises the following steps: Adding fresh sturgeon meat and / or fish eggs to water, breaking the fish wall or homogenizing to obtain a homogenate; The homogenate is centrifuged or plate-and-frame filtered to remove the residue and the extract is collected; The extract is then filtered through a membrane to obtain a liquid sturgeon extract; or further dried to obtain a solid sturgeon extract.

7. The preparation method according to claim 6, wherein The mass ratio of the fish meat and / or fish eggs to water is 1:(0.5-100), preferably 1:(1-30); The preparation temperature of the homogenate is 4-50°C; Preferably, after cell wall breaking or homogenization, further ultrasonication may be performed to obtain a homogenate; Preferably, the cell wall breaking process is performed for 3-30 minutes and 3-20 times. Preferably, the homogenization is carried out at a temperature of 4-30°C, a pressure of 5-60 MPa, and a homogenization frequency of 1-10 times; Preferably, the ultrasonic treatment is carried out at an ultrasonic frequency of 5-50 kHz, an ultrasonic power of 50-1000 W, and an ultrasonic time of 3-60 min; The bag or plate-and-frame filtration conditions used in the centrifugation are 400-600 mesh, and the centrifugation time is 20-30 min; The filter membrane specification used in the membrane filtration is 0.05 μm-5 μm, preferably 0.05-3 μm; The drying method includes one or a combination of two or more of natural drying, spray drying or freeze drying.

8. Use of the sturgeon extract according to any one of claims 1 to 5, or the sturgeon extract prepared by the preparation method according to claim 6 or 7, in the preparation of antioxidant, anti-inflammatory, liver-protecting and / or immune-enhancing products, preferably, the products are medicines, health products or cosmetics.

9. A polypeptide composition, wherein: The polypeptide composition comprises a combination of one or more of the polypeptides shown in any one of SEQ ID NOs: 1-30.

10. Use of the polypeptide composition according to claim 9 in the preparation of products for antioxidant, anti-inflammatory, liver protection and / or immune enhancement, preferably, the product is a medicine, health product or cosmetic.

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