A sturgeon ovary peptide with endocrine function regulating and its preparation method

Abstract

The present application relates to the technical field of functional food, in particular to a sturgeon ovary peptide with endocrine function regulation and a preparation method thereof, which comprises the following steps: taking sturgeon ovary tissue, removing fat and impurities; adding deionized water and sodium ascorbate, homogenizing and vacuum defoaming to obtain a pretreated liquid; adding compound protease to the pretreated liquid for enzymolysis; after the enzymolysis is completed, centrifugal separation is carried out on the supernatant after cooling; the supernatant is separated through an ultrafiltration membrane, and then a low-temperature microwave-assisted oxidation cross-linking reaction is carried out, and hydrogen peroxide and a mixture of naringin / soy isoflavones are added; finally, vacuum freeze-drying is carried out to obtain the sturgeon ovary peptide with endocrine function regulation. In the present application, the microwave-assisted oxidation cross-linking and the incorporation of naringin and soy isoflavones significantly reduce the oxidation loss in the high-temperature drying process, further stabilize the active conformation and enhance the binding affinity to the estrogen receptor.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and more specifically, to a sturgeon ovarian peptide with endocrine-regulating function and its preparation method. Background Technology

[0002] With the deepening research on women's health management and functional peptide products, active peptides derived from fish ovarian tissue have been proven to have multiple physiological functions, such as promoting estrogen balance, improving endocrine disorders, and delaying aging, due to their rich content of small molecule polypeptides, essential amino acids, and hormone precursors. Among them, sturgeon ovaries have the characteristics of high protein content, moderate lipid ratio, and rich active ingredients, and have excellent development potential, making them an ideal source of raw materials for preparing endocrine-regulating active peptides.

[0003] Existing sturgeon ovary peptide preparation processes, when subjected to high-temperature treatment or simulated gastrointestinal digestion conditions, often result in decreased product activity, bitter taste, and insufficient thermal stability, limiting their application in women's nutritional supplements and functional foods. In view of this, we propose a sturgeon ovary peptide with endocrine-regulating function and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to provide a sturgeon ovary peptide with endocrine-regulating function and its preparation method, in order to solve the problem mentioned in the background art that the existing sturgeon ovary peptide preparation process often leads to decreased product activity, bitter taste, and insufficient thermal stability under high temperature treatment or simulated gastrointestinal digestion conditions, which limits its application in women's nutritional supplements and functional foods.

[0005] This invention provides a method for preparing sturgeon ovarian peptides with endocrine-regulating functions, comprising the following steps: S1.1 Take sturgeon ovarian tissue, remove fat and impurities, add deionized water at 8-12 times the weight of ovarian tissue, add sodium ascorbate at 0.5-1.0% of the weight of ovarian tissue, homogenize at 4-6℃ and degas under vacuum to obtain pretreatment solution; S1.2 Add a complex protease to the pretreatment solution, wherein the mass ratio of ovarian tissue to complex protease is 100:1.0-2.5. Stir at 200-300 rpm for 3.0-4.5 h at pH 7.5-8.2 and temperature 38-42℃. After enzymatic hydrolysis, heat to 90-95℃ and incubate for 10-15 min to inactivate the enzyme. After cooling, centrifuge at 8000-10000 rpm for 15 min to separate the supernatant. S1.3 The supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa to collect the peptide solution; the peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction, and a mixture of hydrogen peroxide and naringin / soy isoflavones at a concentration of 0.02-0.05 mol / L was added; then it was freeze-dried under vacuum to obtain sturgeon ovarian peptides with endocrine-regulating function.

[0006] After the reaction is complete, the reaction solution is immediately placed in a vacuum degassing device and degassed for 15-20 minutes at an absolute pressure of 5-15 kPa and a temperature of 20-25°C to ensure that the hydrogen peroxide is completely decomposed and the generated oxygen is removed.

[0007] Preferably, in S1.1, the sturgeon ovarian tissue is taken from a mature ovary 3-5 days before ovulation, and its protein content accounts for 15-20% of the sturgeon ovarian tissue.

[0008] Preferably, in step S1.1, the homogenization speed is 6000-8000 rpm and the homogenization time is 3-5 min.

[0009] Preferably, in step S1.2, the complex protease is composed of trypsin, papain and alkaline protease mixed in an activity ratio of 1:1.0-2.0:0.5-1.5.

[0010] Preferably, in step S1.2, after enzymatic hydrolysis, the solution is decolorized with activated carbon at an amount of 0.3-0.5% of the mass of the hydrolysate, and the decolorization time is 15-20 minutes.

[0011] Preferably, in step S1.3, the ultrafiltration membrane separation pressure is 0.15-0.25 MPa and the temperature is 20-25℃.

[0012] Preferably, in S1.3, the microwave power of the low-temperature microwave-assisted oxidative crosslinking reaction is 300-500W, the temperature is 40-45℃, and the time is 5-8min.

[0013] Preferably, in step S1.3, the amount of the naringin / soy isoflavone mixture added accounts for 0.1-0.3% of the peptide solution mass; The mass ratio of naringin to soy isoflavones in the naringin / soy isoflavone mixture is 1:1.5-3.0.

[0014] Preferably, in step S1.3, the peptide solution is pre-frozen at -20~-30℃ for 4-6 hours before freeze-drying, and then freeze-dried under vacuum at -35~-20℃ for 6-8 hours.

[0015] On the other hand, the present invention provides a sturgeon ovarian peptide with endocrine-regulating function, which is prepared by any of the above-described methods for preparing a sturgeon ovarian peptide with endocrine-regulating function.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, a sturgeon ovary peptide with endocrine-regulating function and its preparation method are disclosed. Microwave-assisted oxidative cross-linking utilizes hydrogen peroxide under mild microwave conditions to induce covalent cross-linking between peptide chains, thereby improving molecular conformational stability and heat resistance, and significantly reducing the oxidative loss of aromatic amino acids during high-temperature drying. Simultaneously, the incorporation of a natural phytoestrogen complex formed by naringin and soy isoflavones can form hydrogen bonds and hydrophobic interactions with the peptide chains at the molecular level, further stabilizing the active conformation and enhancing the binding affinity to estrogen receptors. The synergistic effect of these two factors improves the activity retention rate of the obtained sturgeon ovary peptide, significantly reduces bitterness, and significantly enhances thermal stability and storage stability, thus endowing it with superior endocrine-regulating activity and palatability. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Sturgeon ovary tissue was purchased from Hainan Huatai Biotechnology Co., Ltd.

[0019] Sodium ascorbate (CAS No.: 134-03-2), naringin (CAS No.: 10236-47-2, purity BR, 95%), soy isoflavones (CAS No.: 486-66-8, purity ≥98%), trypsin (CAS No.: 9002-07-7), papain (CAS No.: 9001-73-4), and activated charcoal (CAS No.: 7440-44-0) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] The alkaline protease was purchased from Jiangsu Yihaotian Biotechnology Co., Ltd.

[0021] Example 1: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ through an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0022] Example 2: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.0. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ through an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0023] Example 3: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:2.5. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After the hydrolysis is completed, the enzyme is inactivated by heating to 90 °C for 15 min. After cooling, the supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ through an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0024] Example 4: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ using an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.02mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0025] Example 5: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ through an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.05mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0026] Example 6: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ using an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidative cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.1% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0027] Example 7: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 4 days before ovulation. Its protein content accounts for 17% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 10 times the weight of ovarian tissue and add sodium ascorbate at 0.8% of the weight of ovarian tissue. Homogenize at 7000 rpm for 4 min at 5℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.5:1.0) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 250 rpm for 4.0 h at pH 8.0 and 40 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.4% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 15 min and then cooled. The supernatant is separated by centrifugation at 9000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ using an ultrafiltration membrane with a molecular weight cutoff of 5kDa and a pressure of 0.20MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 400W, a temperature of 40℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:2.0 (0.3% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -25℃ for 5h and then freeze-dried under vacuum at -30℃ for 7h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0028] Example 8: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 3 days before ovulation. Its protein content accounts for 15% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 8 times the weight of ovarian tissue and add sodium ascorbate at 0.5% of the weight of ovarian tissue. Homogenize at 6000 rpm for 3 minutes at 4℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:1.0:0.5) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 200 rpm for 3.0 h at pH 7.5 and 38 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 15 min, and the amount added is 0.3% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 90 °C for 10 min and then cooled. The supernatant is separated by centrifugation at 8000 rpm for 15 min. S1.3 The supernatant was separated at 20℃ through an ultrafiltration membrane with a molecular weight cutoff of 3kDa and a pressure of 0.15MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidative cross-linking reaction at a microwave power of 300W, a temperature of 40℃, and a time of 5min. Hydrogen peroxide with a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones (mass ratio of 1:1.5) at 0.2% of the peptide solution mass were added. Subsequently, the peptide solution was pre-frozen at -20℃ for 4h and then freeze-dried under vacuum at -35℃ for 6h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0029] Example 9: A method for preparing sturgeon ovarian peptides with endocrine-regulating function, comprising the following steps: S1.1 Take mature sturgeon ovarian tissue 5 days before ovulation. Its protein content accounts for 20% of the sturgeon ovarian tissue. Remove fat and impurities. Add deionized water at 12 times the weight of ovarian tissue and add sodium ascorbate at 1.0% of the weight of ovarian tissue. Homogenize at 8000 rpm for 5 minutes at 6℃ and defoam under vacuum to obtain the pretreatment solution. S1.2 Add a complex protease (a mixture of trypsin, papain, and alkaline protease in an activity ratio of 1:2.0:1.5) to the pretreatment solution, wherein the mass ratio of ovarian tissue to the complex protease is 100:1.6. The enzyme is hydrolyzed at 300 rpm for 4.5 h at pH 8.2 and 42 °C. After hydrolysis, the enzyme is decolorized with activated carbon for 20 min, and the amount added is 0.5% of the mass of the hydrolysate. After hydrolysis, the enzyme is inactivated by heating to 95 °C for 15 min. After cooling, the supernatant is separated by centrifugation at 10,000 rpm for 15 min. S1.3 The supernatant was separated at 25℃ using an ultrafiltration membrane with a molecular weight cutoff of 10kDa and a pressure of 0.25MPa to collect the peptide solution. The peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction at a microwave power of 500W, a temperature of 45℃, and a time of 8min. Hydrogen peroxide at a concentration of 0.04mol / L and a mixture of naringin / soy isoflavones at a mass ratio of 1:3.0 (0.2% by weight of the peptide solution) were added. Subsequently, the peptide solution was pre-frozen at -30℃ for 6h and then freeze-dried under vacuum at -20℃ for 8h to obtain sturgeon ovarian peptides with endocrine-regulating functions.

[0030] Determination of endocrine regulatory activity (E2): Rat ovarian granulosa cells were cultured in DMEM / F12 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator. Cells were seeded into 96-well plates (for cell viability assay) and 24-well plates (for hormone assay). A blank control group (containing only culture medium), a model control group (containing 0.1% DMSO), and a positive control group (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) were set up. -8 The culture medium for M estradiol (E2) and the sample group (culture medium containing different concentrations of sturgeon ovarian peptide (e.g., 50, 100, 200 μg / mL)) were used. After culturing for 48 hours, the cell supernatant from the 24-well plates was collected. The E2 concentration was determined using a commercially available rat estradiol (E2) ELISA kit. In microwells coated with anti-E2 antibody, the standard and sample were added sequentially and incubated at room temperature for 1-2 hours. After washing the plate, biotinylated E2 antibody was added and incubated for 1 hour. After washing the plate again, horseradish peroxidase (HRP)-labeled streptavidin was added and incubated for 30 minutes. The substrate TMB was added for color development for 15 minutes, and finally the stop solution was added. The absorbance was measured at 450 nm. The E2 concentration of each sample group was calculated based on the standard curve. The E2 enhancement rate (%) = (E2 concentration of sample group - E2 concentration of model control group) / E2 concentration of model control group × 100%.

[0031] Determination of antioxidant activity (DPPH scavenging rate): Accurately weigh DPPH and prepare a 0.1 mM solution with anhydrous ethanol, storing it in the dark; prepare a 1.0 mg / mL stock solution of sturgeon ovarian peptide with deionized water, and dilute it to a series of concentrations; take 2.0 mL of sample solution and 2.0 mL of DPPH working solution in a test tube, vortex to mix, and react at room temperature in the dark for 30 minutes; using anhydrous ethanol as a reference, measure the absorbance of the reaction solution at a wavelength of 517 nm, and record it as . Simultaneously, the absorbance of 2.0 mL sample solution + 2.0 mL ethanol was measured and recorded as . (Correct the sample's own color); Measure the absorbance of 2.0 mL DPPH working solution + 2.0 mL ethanol, and record it as _____. ; Calculate DPPH clearance rate (%) = [1 - ( - ) / ]×100%.

[0032] Assay for cell proliferation activity (proliferation rate): Ovarian granulosa cells were cultured at 5 × 10⁶ cells per well. 3 -1×10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and pre-cultured at 37°C with 5% CO2 for 24 hours. The old culture medium was discarded, and the experimental groups were the same as those for the endocrine regulatory activity assay. Each group had 5-6 replicates. The plates were cultured for another 44 hours. 10 μL of LCK-8 solution was added to each well. The plates were returned to the incubator and incubated for another 2-4 hours. The absorbance (OD) at 450 nm was measured using a microplate reader. The cell proliferation rate (%) was calculated as follows: - ) / ( - ) × 100%.

[0033] Hormone secretion level (progesterone) determination: Sample source: ovarian granulosa cell supernatant collected in the same batch as the endocrine regulatory activity assay; a commercially available rat progesterone (P) ELISA kit was used, with procedures similar to the E2 kit: sample addition (standard / sample), antibody addition, incubation, washing, enzyme conjugate addition, incubation, washing, substrate addition for color development, termination, reading (450 nm); the progesterone concentration in the sample was directly read from the progesterone standard curve, in ng / mL.

[0034] Determination of thermal stability (activity retention rate): Take an appropriate amount of sturgeon ovarian peptide powder or solution and heat it in a 90℃ water bath or oven for 30 minutes; take an equal amount of the same batch of samples and store them at 4℃ without heating; after the treated group cools to room temperature, measure a certain biological activity (using cell proliferation activity as an indicator) of the control group and the treated group samples respectively; calculate: thermal stability activity retention rate (%) = cell proliferation activity of the treated sample / cell proliferation activity of the untreated control group × 100%.

[0035] Table 1. Performance data of sturgeon ovarian peptides with endocrine-regulating functions. Endocrine regulatory activity (E2 enhancement) Antioxidant activity (DPPH scavenging rate) Cell proliferation activity (proliferation rate) Hormone secretion levels (progesterone) Thermal stability (activity retention rate) Example 1 38.5% 85.2% 132.5% 25.8 ng / mL 92.1% Example 2 25.1% 72.3% 108.3% 18.2 ng / mL 85.6% Example 3 36.8% 84.9% 130.1% 24.9 ng / mL 90.5% Example 4 35.2% 79.5% 126.8% 23.5 ng / mL 88.3% Example 5 37.1% 83.1% 129.2% 24.1 ng / mL 94.7% Example 6 33.7% 81.8% 125.7% 22.6 ng / mL 89.8% Example 7 39.8% 85.6% 135.4% 26.5 ng / mL 93.5% Comparing Examples 1, 2, and 3, it can be seen that the performance indicators of Example 2 (enzyme addition amount 100:1.0) are significantly lower than those of Example 1 (100:1.6) and Example 3 (100:2.5), especially in terms of cell proliferation activity and hormone secretion level, indicating that insufficient enzyme addition leads to incomplete protein hydrolysis and insufficient release of active peptides.

[0036] The performance of Example 1 and Example 3 is similar, indicating that the performance improvement tends to saturate after the enzyme addition reaches 100:1.6. Although further increasing the enzyme amount (Example 3) still maintains good performance, the improvement is limited, indicating that the enzyme addition ratio is close to the optimal balance point between economy and efficiency.

[0037] Comparing Examples 1, 4, and 5, it can be seen that the antioxidant activity (79.5%) and thermal stability (88.3%) of Example 4 (0.02 mol / L hydrogen peroxide) are significantly lower than those of Example 1 (0.04 mol / L) and Example 5 (0.05 mol / L), indicating that insufficient hydrogen peroxide concentration leads to insufficient oxidative cross-linking and fails to effectively stabilize the peptide chain conformation.

[0038] Example 5 exhibited the best thermal stability (94.7%), indicating that moderately increasing the hydrogen peroxide concentration helps to enhance the cross-linking structure between peptide chains, thereby better maintaining activity during heat treatment.

[0039] Comparing Examples 1, 6, and 7, it can be seen that as the amount of naringin / soy isoflavone mixture added increased from 0.1% (Example 6) to 0.3% (Example 7), the endocrine regulatory activity (E2 increase rate increased from 33.7% to 39.8%), cell proliferation activity (increased from 125.7% to 135.4%), and progesterone secretion level (increased from 22.6 ng / mL to 26.5 ng / mL) all showed a dose-dependent increase, indicating that the mixture and sturgeon ovarian peptides have a significant synergistic enhancing effect in regulating endocrine function.

[0040] Based on the above measurements, Example 1 is selected as the optimal example. Comparative Example 1: The difference between this example and Example 1 is that a single alkaline protease is used directly.

[0041] Comparative Example 2: The difference between this example and Example 1 is that the low-temperature microwave-assisted oxidative crosslinking step was not added.

[0042] Comparative Example 3: The difference between this example and Example 1 is that the naringin / soy isoflavone mixture was not added.

[0043] Table 2 Performance data of sturgeon ovarian peptides with endocrine-regulating functions Endocrine regulatory activity (E2 enhancement) Antioxidant activity (DPPH scavenging rate) Cell proliferation activity (proliferation rate) Hormone secretion levels (progesterone) Thermal stability (activity retention rate) Example 1 38.5% 85.2% 132.5% 25.8 ng / mL 92.1% Comparative Example 1 28.3% 75.6% 115.8% 19.4 ng / mL 86.5% Comparative Example 2 35.1% 80.5% 128.9% 24.1 ng / mL 72.3% Comparative Example 3 32.9% 79.8% 121.7% 21.5 ng / mL 90.8% All activity indicators of Comparative Example 1 (single alkaline protease) were significantly lower than those of Example 1 (complex protease), such as endocrine regulation activity (down from 38.5% to 28.3%) and cell proliferation activity (down from 132.5% to 115.8%). This indicates that the complex enzyme can more comprehensively hydrolyze ovarian proteins and release more diverse and more active peptides, while the single enzyme is less hydrolyzed due to the limited number of cleavage sites.

[0044] The thermal stability of Comparative Example 2 (without oxidative crosslinking) decreased significantly (from 92.1% to 72.3%), indicating that the low-temperature microwave-assisted oxidative crosslinking step plays a key role in the stability of peptide chain structure and can effectively enhance its conformational retention ability during heat treatment.

[0045] Comparative Example 3 (without naringin / soy isoflavone mixture) showed significantly lower endocrine regulation activity and cell proliferation activity than Example 1, further confirming that the mixture and sturgeon ovarian peptide have a synergistic effect at the molecular level, jointly enhancing their binding ability to estrogen receptors and cellular regulatory functions.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing sturgeon ovarian peptides with endocrine-regulating function, characterized in that, Includes the following steps: S1.1 Take sturgeon ovarian tissue, remove fat and impurities, add deionized water at 8-12 times the weight of ovarian tissue, add sodium ascorbate at 0.5-1.0% of the weight of ovarian tissue, homogenize at 4-6℃ and degas under vacuum to obtain pretreatment solution; S1.2 Add a complex protease to the pretreatment solution, wherein the mass ratio of ovarian tissue to complex protease is 100:1.0-2.

5. Stir at 200-300 rpm for 3.0-4.5 h at pH 7.5-8.2 and temperature 38-42℃. After enzymatic hydrolysis, heat to 90-95℃ and incubate for 10-15 min to inactivate the enzyme. After cooling, centrifuge at 8000-10000 rpm for 15 min to separate the supernatant. S1.3 The supernatant was separated by an ultrafiltration membrane with a molecular weight cutoff of 3-10 kDa to collect the peptide solution; the peptide solution was subjected to a low-temperature microwave-assisted oxidation cross-linking reaction, and a mixture of hydrogen peroxide and naringin / soy isoflavones at a concentration of 0.02-0.05 mol / L was added; then it was freeze-dried under vacuum to obtain sturgeon ovarian peptides with endocrine-regulating function.

2. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In S1.1, the sturgeon ovarian tissue is taken from a mature ovary 3-5 days before ovulation, and its protein content accounts for 15-20% of the sturgeon ovarian tissue.

3. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In step S1.1, the homogenization speed is 6000-8000 rpm and the homogenization time is 3-5 min.

4. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In S1.2, the complex protease is composed of trypsin, papain and alkaline protease mixed in an activity ratio of 1:1.0-2.0:0.5-1.

5.

5. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In step S1.2, after enzymatic hydrolysis, the solution is decolorized with activated carbon. The amount of activated carbon added is 0.3-0.5% of the mass of the enzymatic hydrolysate, and the decolorization time is 15-20 min.

6. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In step S1.3, the ultrafiltration membrane separation pressure is 0.15-0.25 MPa and the temperature is 20-25℃.

7. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In S1.3, the microwave power of the low-temperature microwave-assisted oxidative crosslinking reaction is 300-500W, the temperature is 40-45℃, and the time is 5-8min.

8. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In step S1.3, the amount of the naringin / soy isoflavone mixture added accounts for 0.1-0.3% of the peptide solution mass; The mass ratio of naringin to soy isoflavones in the naringin / soy isoflavone mixture is 1:1.5-3.

0.

9. The method for preparing sturgeon ovarian peptides with endocrine-regulating function according to claim 1, characterized in that, In step S1.3, the peptide solution is pre-frozen at -20~-30℃ for 4-6 hours before freeze-drying, and then freeze-dried under vacuum at -35~-20℃ for 6-8 hours.

10. A sturgeon ovarian peptide with endocrine-regulating function, characterized in that, It is prepared using the method for preparing sturgeon ovarian peptide with endocrine-regulating function as described in any one of claims 1-9.