Preparation method and application of oyster-derived antioxidant and osteogenesis-promoting bifunctional polypeptide

Abstract

The invention discloses a preparation method and application of an oyster-derived antioxidant and osteogenesis-promoting bifunctional polypeptide, relates to the technical field of biology, and solves the problem of serious insufficiency in research and utilization of oyster bioactive peptides at present, and the preparation method comprises the following steps: step 1, manually shelling to obtain fresh oyster meat, washing with deionized water, and drying to obtain fresh oyster meat; the fresh oyster meat and the extracting solution A are mixed to obtain a mixture A, the mass volume ratio of the fresh oyster meat to the extracting solution A is 1: 3, the extracting solution A is a phosphate buffer solution, the concentration of the phosphate buffer solution is 0.01 mol / L, and the pH of the phosphate buffer solution is 7.2. The enzymolysis efficiency and the in-vitro antioxidant activity are improved through ultrasonic-assisted enzyme hydrolysis, H2O2-induced MC3T3-E1 cell oxidative stress and osteoblast dysfunction can be reversed, and the method is applied to related functional foods for resisting oxidation, promoting bone growth and delaying bone loss.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a preparation method and application of an oyster-derived antioxidant and osteogenic dual-functional polypeptide. Background Art

[0002] Osteoporosis is a common systemic metabolic chronic disease characterized by deterioration of bone density and bone microarchitecture, accompanied by an increased risk of brittle fractures. With the rapid increase in the aging population, the incidence of osteoporosis continues to rise, resulting in an increasing social and medical burden. Oxidative stress plays a vital role in the pathogenesis of osteoporosis. Reactive oxygen species (ROS) can seriously affect the proliferation and differentiation of osteoblasts, disrupt bone balance, and lead to the occurrence of osteoporosis. In view of these challenges, natural active molecules with strong antioxidant and osteogenic activity have received increasing attention and become an ideal alternative for the prevention and treatment of osteoporosis.

[0003] Oysters have important edible and medicinal value and are used in traditional Chinese medicine to supplement calcium and promote bone growth. Protein is one of the most important nutrients in oysters. It is an excellent source for the production of bioactive peptides because it contains essential amino acids, taurine, ornithine and a variety of non-essential amino acids. Studies have shown that peptides extracted from oyster protein have various health benefits, including antioxidant, osteogenic, antibacterial and anti-thrombotic activities. Currently, enzymatic hydrolysis is a common method for producing peptides. However, this method often has disadvantages such as long processing time, low enzyme utilization, low hydrolysis efficiency and low biological activity;

[0004] Ultrasound is an emerging green, clean, efficient, safe and easy-to-operate assisted enzymatic hydrolysis technology, and has been proven to significantly improve the efficiency of enzymatic hydrolysis and the yield and activity of bioactive peptides. The mechanism is that during the ultrasonic treatment, strong fluid dynamic shear, pressure and temperature fluctuations will cause changes in the structure of the substrate, promote the binding of the substrate and the enzyme, thereby improving the enzyme degradation efficiency and the production of bioactive peptides.

[0005] However, the current research and utilization of oyster bioactive peptides are seriously insufficient; therefore, it does not meet the existing needs. In this regard, we propose a preparation method and application of oyster-derived antioxidant and osteogenic dual-functional peptides. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and application of an oyster-derived antioxidant and osteogenic dual-functional polypeptide, so as to solve the problem of the current serious lack of research and utilization of oyster bioactive peptides mentioned in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide, comprising the following steps:

[0008] Step 1: manually peeling the shell to obtain fresh oyster meat, washing it with deionized water, and then mixing it with extract A to obtain mixture A;

[0009] Step 2: The mixture was homogenized at 9,000 × g for 120 min in an ice bath at 4°C. After homogenization, the mixture was centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant A.

[0010] Step 3: freeze-drying the supernatant A to obtain oyster total protein dry powder;

[0011] Step 4: Mix the oyster total protein powder with the extract B, and pre-treat the mixture using an ultrasonic cell crusher, using ultrasonic power T for treatment, ultrasonic frequency of 20 kHz, both the on and off time are set to 2 s, and the total treatment time in a 4°C ice bath is 20 min;

[0012] Step 5: The substrate was enzymatically hydrolyzed with pepsin for 2 h, and then with trypsin for 2 h, both at a hydrolysis temperature of 37°C. The enzyme reaction was inactivated at 100°C for 10 min to obtain supernatant B, which was then centrifuged at 10,000×g for 10 min at 4°C and collected, and freeze-dried to obtain the oyster-derived antioxidant and osteogenic bifunctional polypeptide.

[0013] Preferably, the mass volume ratio of the fresh oyster meat to the extract A is 1:3, the extract A is a phosphate buffer, the concentration of the phosphate buffer is 0.01 mol / L, and the pH of the phosphate buffer is 7.2.

[0014] Preferably, the mass volume ratio of the oyster total protein dry powder to the extract B is 1:7, and the extract B is deionized water.

[0015] Preferably, the ultrasonic power T includes 0W, 50W, 100W, 150W, 200W, 250W and 300W.

[0016] Preferably, the ultrasonic power T is 200W.

[0017] Preferably, the substrate concentrations of pepsin and trypsin are both 5,000 U / gprot.

[0018] The applications of the oyster-derived antioxidant and osteogenic dual-functional polypeptide prepared by the preparation method include preparing products with antioxidant activity, improving free radical scavenging ability, preparing products that promote bone growth, preparing products that delay bone loss, improving the proliferation and differentiation ability of osteoblasts, and preparing products for screening specific antioxidant and osteogenic dual-active polypeptide fragments.

[0019] Preferably, the products include food and biological preparations.

[0020] An osteogenic differentiation medium comprising the oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared by the preparation method, wherein the concentration of the oyster-derived antioxidant and osteogenic bifunctional polypeptide in the medium is 5-125 μg / mL.

[0021] A method for improving antioxidant activity, oxidative stress repair ability and osteogenic differentiation ability of osteoblasts, comprising a preparation method of oyster-derived antioxidant and osteogenic bifunctional polypeptides or culturing osteoblasts using an osteogenic induction differentiation medium.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention discloses for the first time an oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared by ultrasound-assisted enzymatic hydrolysis. Ultrasound-assisted enzymatic hydrolysis improves the enzymatic hydrolysis efficiency and its in vitro antioxidant activity, which can reverse H 2 O 2 Induced oxidative stress and osteoblast dysfunction in MC3T3-E1 cells, wherein oyster-derived antioxidant and osteogenic bifunctional peptides can be used as beneficial food additives in functional foods related to antioxidant, bone growth promotion and bone loss retardation;

[0024] 2. No organic reagent needs to be added during the preparation process of the present invention, and the preparation method is simple and the ultrasonic treatment conditions are mild. As a common seafood, oyster raw materials are easy to obtain and rich in protein, which can meet the needs of mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The flowchart of the preparation method of the oyster-derived antioxidant and osteogenic dual-functional polypeptide of the present invention;

[0026] Figure 2 This is a schematic diagram of the hydrolysis degree of oyster-derived antioxidant and osteogenic dual-functional peptides;

[0027] Figure 3 This is a schematic diagram of the soluble peptide concentration of the oyster-derived antioxidant and osteogenic bifunctional polypeptide;

[0028] Figure 4 A schematic diagram of the hydroxyl radical scavenging rate of the in vitro antioxidant capacity of oyster-derived antioxidant and osteogenic bifunctional peptides;

[0029] Figure 5 ABTS statistics for the in vitro antioxidant capacity of oyster-derived antioxidant and osteogenic bifunctional peptides + Schematic diagram of free radical scavenging rate;

[0030] Figure 6 The result statistical diagram of the molecular weight distribution of oyster-derived antioxidant and osteogenic bifunctional polypeptides;

[0031] Figure 7 This is a statistical chart showing the effects of 200W ultrasound pretreatment of oyster-derived antioxidant and osteogenic bifunctional peptides on the survival rate of H2O2-induced osteogenic MC3T3-E1 cells;

[0032] Figure 8 Representative images of ROS staining of oyster-derived antioxidant and osteogenic bifunctional peptides on H2O2-induced osteogenic MC3T3-E1 cells pretreated with 200W ultrasound;

[0033] Fig. 9 This is a statistical chart showing the quantitative analysis of the ROS fluorescence signal of MC3T3-E1 osteogenic cells induced by H2O2 by oyster-derived antioxidant and osteogenic bifunctional peptides pretreated with 200W ultrasound;

[0034] Fig.10 Schematic diagram of the quantitative effect of oyster-derived antioxidant and osteogenic bifunctional peptides pretreated with 200W ultrasound on the ALP activity of H2O2-induced osteogenic MC3T3-E1 cells;

[0035] Fig.11 A representative schematic diagram of Alizarin Red staining;

[0036] Fig.12 This is a statistical chart of the quantitative analysis results of Alizarin red staining. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0038] See also Figures 1 to 12 , the present invention provides four embodiments:

[0039] Example 1: A method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide, comprising the following steps:

[0040] Step 1: manually peeling the shell to obtain fresh oyster meat, washing it with deionized water, and then mixing it with extract A to obtain a mixture A, wherein the mass volume ratio of the fresh oyster meat to the extract A is 1:3, the extract A is a phosphate buffer, the concentration of the phosphate buffer is 0.01 mol / L, and the pH of the phosphate buffer is 7.2;

[0041] Step 2: The mixture was homogenized at 9,000 × g for 120 min in an ice bath at 4°C. After homogenization, the mixture was centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant A.

[0042] Step 3: freeze-drying the supernatant A to obtain oyster total protein dry powder;

[0043] Step 4: Mix the oyster total protein dry powder with the extract B, the mass volume ratio of the oyster total protein dry powder to the extract B is 1:7, the extract B is deionized water, and the mixture is pretreated with an ultrasonic cell crusher, and treated with ultrasonic power T, the ultrasonic power T includes 0W, 50W, 100W, 150W, 200W, 250W and 300W, and different ultrasonic powers T are used for treatment, the ultrasonic frequency is 20kHz, the on and off time are set to 2s, and the total treatment time in a 4°C ice bath is 20min;

[0044] Step 5: The substrate was enzymatically hydrolyzed with pepsin for 2 h, and then with trypsin for 2 h. The substrate concentrations of pepsin and trypsin were both 5,000 U / gprot, and the enzymatic hydrolysis temperature was 37°C. The enzyme reaction was inactivated at 100°C for 10 min to obtain supernatant B. The supernatant B was then centrifuged at 10,000 × g for 10 min at 4°C, collected, and freeze-dried to obtain the oyster-derived antioxidant and osteogenic bifunctional polypeptide.

[0045] Example 2: An application of the above-mentioned oyster-derived antioxidant and osteogenic dual-functional polypeptide, including preparing products with antioxidant activity, improving free radical scavenging ability, preparing products that promote bone growth, preparing products that delay bone loss, improving the proliferation and differentiation ability of osteoblasts, and preparing products for screening specific antioxidant and osteogenic dual-active polypeptide fragments, and the products include food and biological preparations.

[0046] Example 3: An osteogenic differentiation medium of the oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared by the above-mentioned preparation method, wherein the concentration of the oyster-derived antioxidant and osteogenic bifunctional polypeptide in the medium is 5-125 μg / mL.

[0047] Example 4: A method for improving antioxidant activity, oxidative stress repair ability and osteogenic differentiation ability of osteoblasts, comprising the steps of preparing the oyster-derived antioxidant and osteogenic bifunctional polypeptide in Example 1 and culturing osteoblasts using the culture medium in Example 3.

[0048] The oyster-derived antioxidant and osteogenic bifunctional polypeptides prepared in Example 1 and pretreated with different ultrasonic powers were characterized in various ways:

[0049] Yield of oyster-derived antioxidant and osteogenic bifunctional polypeptide: The protein concentration of the oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared in Example 1 was determined by the Kjeldahl method, and the yield of the oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared in Example 1 was calculated according to the following formula: yield (%) = 100% × weight of freeze-dried powder after ultrasound-assisted enzymatic hydrolysis / weight of oyster total protein dry powder;

[0050] The results are shown in Table 1 below. The peptide yield increases with the increase of power within the ultrasonic power range of 0 to 200 W, and the peptide yield is the highest at 200 W, indicating that within a certain ultrasonic power range, the increase of ultrasonic pretreatment power is conducive to the unfolding of protein structure and improves enzymatic hydrolysis efficiency;

[0051] Table 1

[0052]

[0053] Hydrolysis degree and soluble peptide concentration of oyster-derived antioxidant and osteogenic bifunctional polypeptide: 100 μL of the oyster-derived antioxidant and osteogenic bifunctional polypeptide sample solution prepared in Example 1 was mixed with 2 mL of freshly prepared OPA reagent, and then the absorbance was measured at a wavelength of 340 nm using an ultraviolet spectrophotometer;

[0054] The oyster-derived antioxidant and osteogenic bifunctional peptide prepared in Example 1 was mixed with 10% trichloroacetic acid (TCA) at a mass volume ratio (m:v) of 1:1, and then centrifuged at 3,000 rpm for 10 min. The peptide content in the supernatant was then quantified using the Lowry method;

[0055] The results are as shown in the instruction manual Figure 2 and Figure 3 As shown in the figure, within the ultrasonic power range of 0 to 300 W, the hydrolysis degree and soluble peptide content increased with the increase of ultrasonic power, reaching the maximum value at 200 W, and slightly decreased after exceeding 200 W. Specifically, the hydrolysis degree of the polypeptide increased from 12.94% at 0 W to 22.61% at 200 W, and its soluble peptide content increased from 22.53 g / 100 g dry weight at 0 W to 32.09 g / 100 g dry weight at 200 W, indicating that appropriate ultrasonic treatment can significantly improve protein hydrolysis and the release of soluble peptides;

[0056] In vitro antioxidant activity assay of oyster-derived antioxidant and osteogenic bifunctional peptides: Hydroxyl radical scavenging activity: 10 mg of sample was dissolved in 1 mL of deionized water, then mixed with 1 mL of 8 mM ferric sulfate and 1 mL of 3 mM salicylic acid, and then 250 μL of 20 mM H 2 O 2, a control group was prepared using a reaction mixture without a sample, and then the absorbance was recorded at a wavelength of 536 nm, and the hydroxyl radical scavenging activity was calculated using the following formula: Hydroxyl radical scavenging rate (%) = 100% × (A control – A sample) / A control. ABTS + radical scavenging activity: According to the instructions provided by the ABTS detection kit (Beyotime, S0121): 10 mg / mL sample solution was mixed with ABTS working solution, reacted for 10 min, and then the absorbance was measured at a wavelength of 414 nm. A control group was prepared using a reaction mixture without a sample, and the results were calculated using the following formula: ABTS + radical scavenging rate (%) = (A control – A sample) / A control × 100%;

[0057] As the instruction manual Figure 4 As shown, with the increase of ultrasonic power, the ability of the polypeptide described in Example 1 to remove OH gradually increases, and the effect is most obvious at 200W (39.86%), and slightly decreases when it exceeds 200W.

[0058] As the instruction manual Figure 5 As shown, similarly, in the ABTS+ scavenging test, the ability of the peptide to scavenge ABTS+ increased from 36.17% at 0 W to 55.20% at an ultrasonic power of 200 W, and then decreased slightly. These results indicate that appropriate ultrasonic treatment can improve the antioxidant capacity of oyster-derived antioxidant and osteogenic bifunctional peptides;

[0059] Based on the above results, the components with 0 to 200 W were selected for subsequent analysis.

[0060] The molecular weight distribution of the oyster-derived antioxidant and osteogenic bifunctional polypeptide pretreated with 0-200W ultrasonic power obtained in Example 2 was further determined. The filtered oyster-derived antioxidant and osteogenic bifunctional polypeptide (2 mg / mL) was separated using a semi-preparative liquid chromatography system equipped with a reversed phase column (TSKgelG2000SWXL, 7.8×300 mm). Isocratic elution was performed using a 45% acetonitrile solution containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 0.5 mL / min. The absorbance monitoring wavelength was 214 nm, and the sample injection volume was 1 mL. The mass standard curve was calibrated using cytochrome C (12,327.8 Da), aprotinin (6,511.4 Da), vitamin B12 (1,355.4 Da), glutathione (307.3 Da) and glycine (75.1 Da);

[0061] As the instruction manual Figure 6 As shown in Figure 2, the molecular weight distribution of oyster-derived antioxidant and osteogenic bifunctional peptides is divided into five ranges: <500Da, 500-1,000Da, 1,000-3,500Da, 3,500-8,000Da.

[0062] and >8,000Da. As the ultrasonic treatment power increased, the content of small molecular weight polypeptides (<3,500Da) increased, indicating that ultrasonic treatment significantly changed the molecular weight distribution of active peptides, and the content of small molecular weight polypeptides (<3,500Da) in the 200W component was the highest;

[0063] The oyster-derived antioxidant and osteogenic dual-functional peptide obtained in Example 3 was pretreated with 200 W ultrasonic power, and its effect on H 2 O 2 Repair effects of induced oxidative stress on osteoblasts:

[0064] MC3T3-E1 cell viability protection: 5×103 cells / well of MC3T3-E1 cells were inoculated into a 96-well plate and cultured at 37°C for 24 h. 2 O 2 The cells were treated for 6 h, and then different concentrations (5 μg / mL, 25 μg / mL, 125 μg / mL) of the polypeptide described in Example 3 were added, and then cultured for another 24 h. The viability of MC3T3-E1 cells was determined using a CCK-8 kit (Beyotime, C0038).

[0065] As the instruction manual Figure 7 As shown, compared with the blank group, 2 O 2 The cell survival rate of the treated model group decreased to 50.78%. However, the treatment with the polypeptide described in Example 3 can restore cell viability in a dose-dependent manner, and the survival rates increased to 55.15%, 61.87% and 65.72% after treatment with 5, 25 and 125 μg / mL doses, respectively.

[0066] Intracellular ROS level of MC3T3-E1: The intracellular ROS level was assessed using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe according to the instructions of the ROS detection kit (Beyotime, S0033S): the cell culture medium was replaced with 5 μM DCFH-DA, and the cells were incubated at 37°C for 20 min. The extracellular DCFH-DA was then removed and the cells were rinsed with PBS. Fluorescence images were then captured using an inverted fluorescence microscope (Leica DMI4000B, Wetzlar) with an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The fluorescence intensity was quantified using ImageJ.

[0067] As the instruction manual Figure 8 and Fig. 9 As shown, the treatment with the polypeptide described in Example 3 can dose-dependently reduce the expression of H in MC3T3-E1 cells. 2 O2 Compared with the model group, the fluorescence intensity of ROS in the induced cells decreased by 12.85% (5 μg / mL), 32.27% (25 μg / mL) and 45.35%, respectively.

[0068] (125 μg / mL).

[0069] The above results all indicate that oyster-derived antioxidant and osteogenic bifunctional peptides pretreated with 200W ultrasonic power can effectively alleviate H 2 O 2 Induced oxidative stress levels in osteoblasts MC3T3-E1.

[0070] The oyster-derived antioxidant and osteogenic dual-functional polypeptide pretreated with 200 W ultrasonic power used in Example 4 was used to investigate its effect on H 2 O 2 Induced osteogenic differentiation dysfunction repair effect:

[0071] Alkaline phosphatase (ALP) activity assay: MC3T3-E1 cells (5×104 cells / mL) were inoculated into 12-well plates and cultured for 24 h. Then, the culture medium prepared by the osteogenic differentiation kit (ZQXZBIO) (including the polypeptide used in Example 4 and 400 μM H 2 O 2 ) for osteogenic induction and differentiation. After 7 days of treatment, cells were rinsed with PBS, ALP activity was measured using an ALP kit (Beyotime) according to the manufacturer's instructions, absorbance was measured at a wavelength of 520 nm to quantify enzyme activity, and enzyme activity was calculated based on protein concentration detected using a BCA protein assay kit (Beyotime). ALP activity was normalized to total protein content.

[0072] As the instruction manual Fig.10 As shown, H 2 O 2 Treatment significantly reduced the activity level of ALP, indicating that H 2 O 2 However, the polypeptide used in Example 4 can effectively improve H in a dose-dependent manner. 2 O 2 The induced ALP level decreased, and compared with the model group, the doses of 5, 25, and 125 μg / mL increased the ALP activity by 0.10-fold, 0.20-fold, and 0.36-fold, respectively.

[0073] Alizarin red S staining: MC3T3-E1 cells (1×105 cells / mL) were inoculated into 12-well plates and cultured for 24 h. Then, the culture medium prepared by the osteogenic differentiation kit (ZQXZBIO) (including the polypeptide used in Example 4 and 400 μM H 2 O 2 ) for osteogenic induction and differentiation. After 21 days of treatment, the cells were stained with Alizarin Red S in the osteoblast differentiation kit. After staining, the cells were rinsed with PBS to remove excess dye, and the mineralized nodules were observed under a microscope and then quantified using 10% cetylpyridinium chloride at a wavelength of 562 nm.

[0074] As the instruction manual Fig.11 and Fig.12 As shown, H 2 O 2 Treatment with H significantly reduced the level of mineralized nodules in MC3T3-E1 cells. 2 O 2 Compared with the model group treated with , the polypeptide used in Example 4 dose-dependently increased the formation of calcium nodules in MC3T3-E1 cells. When the concentration of the polypeptide was 125 μg / mL (1.38 times that of the model group), the promoting effect on mineralization was the most obvious.

[0075] The above results show that the oyster-derived antioxidant and osteogenic dual-functional polypeptide pretreated with 200W ultrasonic power used in Example 4 can effectively improve H 2 O 2 The induced osteogenic dysfunction of MC3T3-E1 cells indicated its potential osteogenic activity.

[0076] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0077] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide, characterized in that: The steps include: Step 1: manually peeling the shell to obtain fresh oyster meat, washing it with deionized water, and then mixing it with extract A to obtain mixture A; Step 2: The mixture was homogenized at 9,000 × g for 120 min in an ice bath at 4°C. After homogenization, the mixture was centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant A. Step 3: freeze-drying the supernatant A to obtain oyster total protein dry powder; Step 4: Mix the oyster total protein powder with the extract B, and pre-treat the mixture using an ultrasonic cell crusher, using ultrasonic power T for treatment, ultrasonic frequency of 20 kHz, both the on and off time are set to 2 s, and the total treatment time in a 4°C ice bath is 20 min; Step 5: The substrate was enzymatically hydrolyzed with pepsin for 2 h, and then with trypsin for 2 h, both at a hydrolysis temperature of 37°C. The enzyme reaction was inactivated at 100°C for 10 min to obtain supernatant B, which was then centrifuged at 10,000×g for 10 min at 4°C and collected, and freeze-dried to obtain the oyster-derived antioxidant and osteogenic bifunctional polypeptide.

2. The method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide according to claim 1, characterized in that: The mass volume ratio of the fresh oyster meat to the extract A is 1:3, the extract A is a phosphate buffer, the concentration of the phosphate buffer is 0.01 mol / L, and the pH of the phosphate buffer is 7.

2.

3. The method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide according to claim 2, characterized in that: The mass volume ratio of the oyster total protein dry powder to the extract B is 1:7, and the extract B is deionized water.

4. The method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide according to claim 3, characterized in that: The ultrasonic power T includes 0W, 50W, 100W, 150W, 200W, 250W and 300W.

5. The method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide according to claim 4, characterized in that: The ultrasonic power T is 200W.

6. The method for preparing an oyster-derived antioxidant and osteogenic dual-functional polypeptide according to claim 3, characterized in that: The substrate concentrations of pepsin and trypsin were both 5,000 U / gprot.

7. Use of the oyster-derived antioxidant and osteogenic dual-functional polypeptide prepared by the preparation method according to any one of claims 1 to 6, characterized in that: It includes preparing products with antioxidant activity, improving free radical scavenging ability, preparing products that promote bone growth, preparing products that delay bone loss, improving the proliferation and differentiation ability of osteoblasts, and preparing products for screening specific antioxidant and osteogenic dual-active polypeptide fragments.

8. The use according to claim 7, characterized in that: The products include food and biologics.

9. An osteogenic differentiation medium comprising an oyster-derived antioxidant and osteogenic bifunctional polypeptide prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The concentration of the oyster-derived antioxidant and osteogenic bifunctional polypeptide in the culture medium is 5-125 μg / mL.

10. A method for improving antioxidant activity, oxidative stress repair ability and osteogenic differentiation ability of osteoblasts, comprising the preparation method of the oyster-derived antioxidant and osteogenic bifunctional polypeptide according to any one of claims 1 to 6 or culturing osteoblasts using the osteogenic differentiation medium according to claim 9.

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