Extraction process of mussel protein for skin repair

Abstract

The invention relates to the technical field of biological extraction, in particular to an extraction process of mussel protein for skin repair, and the extraction process of the mussel protein comprises the following steps: step 1, taking fresh mytilus edulis byssus glands, washing, and crushing at low temperature to obtain a pre-treated substance; step 2, adding alkaline protease into the pretreated substance, carrying out oscillation treatment, and centrifuging to obtain supernate a; step 3, adjusting the supernatant a, adding neutral protease, performing ultrasonic-assisted synchronous microwave radiation, adding tyrosinase, controlling the throughput, and performing enzymolysis to obtain enzymatic hydrolysate; and step 4, extracting the enzymatic hydrolysate by adopting supercritical CO2, carrying out ultrafiltration concentration and ion exchange chromatography technologies, collecting eluent, and freeze-drying to obtain the mussel protein for skin repair. The prepared mussel protein is high in yield, high in purity and low in impurity content, and the endotoxin content is effectively controlled, so that the biological safety of the product is ensured, free radicals can be effectively removed, and the oxidation reaction process is delayed.

Description

Technical Field

[0001] This application relates to the field of biological extraction technology. More specifically, it relates to an extraction process for mussel protein used for skin repair. Background Art

[0002] Most of the current skin repair products on the market rely on chemically synthesized ingredients or plant extracts. Chemically synthesized ingredients often have the problem of strong irritation, which may cause adverse reactions such as skin allergies and redness. Although plant extracts are relatively mild, they have certain limitations in terms of repair effects and are difficult to meet people's needs for efficient skin repair. Therefore, finding a skin repair ingredient that is both safe and efficient has become an important research direction.

[0003] As a natural marine bioactive protein, mussel protein has received extensive attention in recent years due to its unique structure and function. Mussel protein is mainly derived from the byssus gland of mussels. Its molecular structure contains abundant polybasic groups (3,4-dihydroxy-L-phenylalanine). Due to its unique biocompatibility, antioxidant property, and adhesiveness, mussel protein has significant potential in the field of skin repair.

[0004] However, the existing mussel protein extraction processes mostly rely on methods such as salting out, multiple centrifugations, and chromatographic purification. The steps are lengthy and the equipment investment is high, resulting in a long extraction cycle and low protein recovery rate. High-temperature extraction and strong acid / alkali treatment are likely to damage the polybasic groups and lysine positive charge structure of mussel mucin, leading to a significant decline in the functions of promoting cell adhesion and antioxidant property. Based on the above statements, this application provides an extraction process that is efficient, environmentally friendly, and can retain the polybasic groups and biological activity of mussel protein for the field of skin repair. Summary of the Invention

[0005] To solve the technical problems mentioned in the background art, this application provides an extraction process for mussel protein used for skin repair.

[0006] An extraction process for mussel protein used for skin repair adopts the following technical scheme: Step 1: Take fresh byssus glands of Mytilus edulis, rinse them with physiological saline 3 - 5 times, and crush them at low temperature to a particle size of 1 - 2 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid - disodium hydrogen phosphate buffer solution with a pH of 7.5 - 8.0 according to a liquid - solid ratio of 3 - 5:1, add alkaline protease, and oscillate at a rotation speed of 100 - 200 rpm and a temperature of 40 - 45 °C for 2 - 3 h, then centrifuge and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of the supernatant a to 7.0 - 7.2. First, add neutral protease, and perform ultrasonic-assisted synchronous microwave radiation. React at a temperature of 42 - 48 °C for 2 - 3 h, then adjust the pH to 6.5 - 6.8, and then add tyrosinase. Control the ventilation volume and enzymatically hydrolyze for 3 - 4 h at 25 - 37 °C to obtain the enzymatically hydrolyzed solution. Step 4: Use supercritical CO2 extraction to remove lipids from the enzymatically hydrolyzed solution. After ultrafiltration concentration and ion exchange chromatography technology, collect the eluate with a volume of 8 - 10 times the column volume, and freeze-dry to obtain the mussel protein for skin repair.

[0007] Preferably, in the step 2, the mass ratio of the alkaline protease to the pretreatment is 0.5 - 2:100 - 150.

[0008] Preferably, in the step 3, the mass ratio of the neutral protease, tyrosinase to the pretreatment is 0.5 - 1.2:0.5 - 0.8:120 - 150.

[0009] Preferably, in the ultrasonic-assisted synchronous microwave radiation in the step 3, the specific conditions of ultrasonic are: ultrasonic power 200 - 400 W, ultrasonic frequency 30 - 40 kHz, working in pulse mode for 20 - 30 s, and intermittent for 10 - 15 s; the specific conditions of microwave radiation are: segmented control, with a power of 400 - 600 W in the first 15 - 30 min, and then the power is adjusted to 200 - 300 W.

[0010] Preferably, the ventilation volume in the step 3 is 0.5 - 2 vvm.

[0011] Preferably, in the step 4, the supercritical CO2 extraction pressure is 20 - 25 MPa, the extraction temperature is 35 - 40 °C, the extraction time is 1 - 2 h, and the CO2 flow rate is 5 - 15 L / min.

[0012] Preferably, the ultrafiltration concentration conditions in the step 4 are: the molecular weight cut-off of the ultrafiltration membrane is 10 - 30 kDa, the pressure is 0.1 - 0.3 MPa, the temperature is 4 - 25 °C, the pH is 6.5 - 7.5, the concentration multiple is 5 - 10 times, and the cross-flow velocity is 1 - 3 m / s.

[0013] Preferably, the ultrafiltration membrane is one of polysulfone ultrafiltration membrane, polyethersulfone ultrafiltration membrane, and polyacrylonitrile ultrafiltration membrane.

[0014] Preferably, the ion exchange chromatography conditions in the step 4 are: the packing is CM Sepharose FF cation exchange resin, the buffer solution is acetic acid buffer solution with a pH of 4 - 6, the eluent is acetic acid buffer solution with a pH of 4 - 6 containing 0 - 1 mol / L NaCl, the inner diameter of the column is 1 - 2 cm, the column length is 20 - 50 cm, the flow rate is 1 - 3 mL / min, and the operating temperature is 25 - 30 °C.

[0015] In summary, the present application has the following beneficial effects: In the mussel protein extraction process of the present application, alkaline protease, neutral protease, and tyrosinase are sequentially used for treatment. The enzymatic hydrolysis efficiency is high, resulting in a high yield of the product. During the enzymatic hydrolysis process, alkaline protease is first used to specifically hydrolyze the peptide bonds formed by basic amino acids, which can effectively destroy the complex protein structure of the mussel byssus gland and preliminarily degrade macromolecular heteroproteins. Then, neutral protease acts on the peptide bonds composed of neutral amino acids to further decompose the protein fragments that are not completely hydrolyzed by alkaline protease, expanding the scope of enzymatic hydrolysis and increasing the dissolution rate and degree of hydrolysis of proteins. Finally, tyrosinase can catalyze the oxidation of tyrosine residues of tyrosine to generate dopa (DOPA) and dopaquinone, which are key functional groups in mussel byssus proteins. Mussel byssus proteins are rich in dopa compounds and have extremely strong adhesion and biocompatibility. They can tightly bind to skin tissues in a humid environment and form hydrogen bonds or covalent bonds with collagen, fibronectin, etc. in the skin, enhancing the adhesion ability and biological activity of the proteins. The dopa compounds catalyzed by tyrosinase can further participate in the cross-linking between protein molecules (such as forming isopeptide bonds or quinone-amino cross-links), optimizing the spatial structure of the proteins and endowing them with stronger mechanical properties and stability. Moreover, after treatment with tyrosinase, the introduction of charged dopaquinone improves the selectivity and purity of chromatographic separation, and the functional structure of mussel proteins is retained through mild enzymatic hydrolysis, avoiding the destruction of their skin repair activities caused by over-hydrolysis, such as promoting collagen synthesis, antioxidant, and anti-inflammatory effects. The three enzymes play a synergistic role. By stepwise adjusting the pH and temperature, they enzymatically hydrolyze layer by layer for different peptide bonds and protein structures, not only improving the protein extraction efficiency but also avoiding the limitations of single-enzyme treatment.

[0016] In the extraction process of mussel proteins of the present application, the ultrasonic-assisted synchronous microwave radiation technology is used to synergistically enhance the enzymatic hydrolysis efficiency and improve the protein release rate. The cavitation bubbles generated by high-frequency vibration rupture, which can destroy the cell structure of the Mytilus edulis byssus gland, accelerate the breaking of cell walls / membranes, make the mussel proteins inside the cells more easily released into the solution, and at the same time increase the contact area between the enzyme and the substrate. Microwave thermal effect and non-thermal effect: The high-frequency electromagnetic waves of microwaves can quickly and uniformly heat the system, and then reduce the power to maintain a constant temperature, avoiding enzyme inactivation caused by local overheating; it can also promote the polar movement of molecules, accelerate the collision frequency between the substrate and the enzyme, significantly shorten the enzymatic hydrolysis time, and improve the enzymatic hydrolysis reaction rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1SDS-PAGE pattern of mussel protein (3 mg / mL) prepared in Example 1; among them, lane 1 is protein Marker; lane 2 is supernatant a with a sample loading volume of 3 μL; lane 3 is the enzymolysis solution with a sample loading volume of 3 μL; lane 3 is the eluate with a sample loading volume of 3 μL; lane 4 is the reconstituted mussel protein with a sample loading volume of 3 μL. Detailed implementation mode

[0018] The following further elaborates on this application in conjunction with examples.

[0019] The alkaline protease used in the examples and comparative examples of the present invention was purchased from Hebei Chuangzhiyuan Biotechnology Co., Ltd.; neutral protease (enzyme activity: 50,000) was purchased from Jiangsu Yuanyuan Biotechnology Co., Ltd.; tyrosinase (product number: GA7589) was purchased from Hubei Guangao Biotechnology Co., Ltd.; CM Sepharose FF cation exchange resin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] Examples 1-3 provide an extraction process for mussel protein for skin repair. Example 1

[0021] An extraction process for mussel protein for skin repair, including the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 according to a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100. Oscillate at a rotation speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a rotation speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0. First add neutral protease, and perform ultrasonic-assisted synchronous microwave radiation. React at a temperature of 42 °C for 2 h, adjust the pH to 6.5, then add tyrosinase, and control the ventilation volume to 0.5 vvm at 25 °C for enzymatic hydrolysis for 3 h to obtain an enzymolysis solution. Among them, the mass ratio of neutral protease, tyrosinase to pretreatment product is 0.5:0.5:120. The specific conditions of ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s, and intermittent for 10 s; the specific conditions of microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymolysis solution by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 8 column volumes of eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluate is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C. Example 2

[0022] An extraction process of mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse 4 times with physiological saline, and crush them at low temperature to a particle size of 1.5 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.8 according to a liquid-solid ratio of 4:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 1:125, oscillate at a rotation speed of 150 rpm at a temperature of 42 °C for 2.5 h, centrifuge at a rotation speed of 9000 rpm for 12 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.1, first add neutral protease, perform ultrasonic-assisted synchronous microwave radiation, react at a temperature of 45 °C for 2.5 h, adjust the pH to 6.6, then add tyrosinase, and control the ventilation volume at 1 vvm at 32 °C for enzymatic hydrolysis for 3.5 h to obtain an enzymolysis solution. Among them, the mass ratio of neutral protease, tyrosinase to the pretreatment product is 0.8:0.6:135. The specific conditions of ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 300 W, ultrasonic frequency of 35 kHz, working in pulse mode for 25 s and intermittent for 12 s; the specific conditions of microwave radiation are segmented control, with a power of 500 W in the first 20 min and then adjusted to 250 W; Step 4: Remove lipids from the enzymolysis solution by supercritical CO2 extraction, concentrate it by ultrafiltration, and use ion exchange chromatography technology to collect the eluate with a volume of 9 times the column volume, and then freeze-dry it to obtain the mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 22 MPa, the extraction temperature is 38 °C, the extraction time is 1.5 h, and the CO2 flow rate is 10 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane, the molecular weight cut-off of the ultrafiltration membrane is 20 kDa, the pressure is 0.2 MPa, the temperature is 20 °C, the pH is 7.0, the concentration multiple is 8 times, and the cross-flow velocity of the ultrafiltration membrane is 2 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 5, the eluate is an acetic acid buffer solution with a pH of 5 containing 0.5 mol / L NaCl, the inner diameter of the column is 1.5 cm, the column length is 35 cm, the flow rate is 2 mL / min, and the operating temperature is 28 °C. Example 3

[0023] An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 5 times with physiological saline, and crush them at low temperature to a particle size of 2 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 8.0 according to a liquid-solid ratio of 5:1, add alkaline protease according to a mass ratio of alkaline protease to the pretreatment product of 2:150, oscillate at a speed of 200 rpm at a temperature of 45 °C for 3 h, centrifuge at a speed of 10,000 rpm for 15 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.2, first add neutral protease, perform ultrasonic-assisted synchronous microwave radiation, react at a temperature of 48 °C for 3 h, adjust the pH to 6.8, then add tyrosinase, and control the ventilation volume at 2 vvm at 37 °C for 4 h to obtain an enzymolysis solution. Among them, the mass ratio of neutral protease, tyrosinase to the pretreatment product is 1.2:0.8:150. The specific conditions of ultrasonic in the ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 400 W, ultrasonic frequency of 40 kHz, working in pulse mode for 30 s, and intermittent for 15 s; the specific conditions of microwave radiation are controlled in segments, with a power of 600 W in the first 30 min and then adjusted to 300 W; Step 4: Remove lipids from the enzymolysis solution by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 10 column volumes of eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 25 MPa, the extraction temperature is 40 °C, the extraction time is 2 h, and the CO2 flow rate is 15 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 30 kDa, the pressure is 0.3 MPa, the temperature is 25 °C, the pH is 7.5, the concentration factor is 10 times, and the cross-flow velocity of the ultrafiltration membrane is 3 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetate buffer solution with a pH of 6, the eluate is an acetate buffer solution with a pH of 6 containing 1.0 mol / L NaCl, the column inner diameter is 2 cm, the column length is 50 cm, the flow rate is 3 mL / min, and the operating temperature is 30 °C.

[0024] Comparative Example 1 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, oscillate at a rotation speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a rotation speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, first add neutral protease, perform ultrasonic-assisted synchronous microwave radiation, react at a temperature of 42 °C for 2 h, adjust the pH to 6.5, then add tyrosinase, control the ventilation volume at 0.5 vvm at 25 °C, and enzymolyze for 3 h to obtain an enzymolysis solution. Among them, the mass ratio of neutral protease, tyrosinase to the pretreatment product is 0.5:0.5:120. The specific conditions of ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s and intermittent for 10 s; the specific conditions of microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 8 column volumes of the eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluate is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0025] Comparative Example 2 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100, oscillate at a speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, perform ultrasonic-assisted synchronous microwave radiation at a temperature of 42 °C for 2 h, adjust the pH to 6.5, add tyrosinase, and control the aeration rate at 0.5 vvm at 25 °C for enzymatic hydrolysis for 3 h to obtain an enzymatic hydrolysate. Among them, the mass ratio of tyrosinase to the pretreatment product is 1:120. The specific conditions of ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s, and intermittent for 10 s; the specific conditions of microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect the eluate with a volume of 8 column volumes and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration multiple is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the conditions for ion exchange chromatography are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluent is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0026] Comparative Example 3 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 according to a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100, and oscillate at a rotation speed of 100 rpm at a temperature of 40 °C for 2 h. Centrifuge at a rotation speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, add neutral protease, and perform ultrasonic-assisted synchronous microwave radiation at a temperature of 42 °C for 5 h to obtain an enzymatic hydrolysate. Among them, the mass ratio of neutral protease to pretreatment product is 1:120. The specific conditions for ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s and intermittent for 10 s; the specific conditions for microwave radiation are controlled in sections, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 8 column volumes of the eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the conditions for ion exchange chromatography are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetate buffer solution with a pH of 4, the eluate is an acetate buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0027] Comparative Example 4 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 according to a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100. Oscillate at a speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, add neutral protease and tyrosinase, and perform ultrasonic-assisted synchronous microwave radiation. At a temperature of 42 °C, control the ventilation volume to 0.5 vvm and enzymatically hydrolyze for 5 h to obtain an enzymatic hydrolysate. Among them, the mass ratio of neutral protease, tyrosinase to pretreatment product is 0.5:0.5:120. The specific conditions for ultrasonic-assisted synchronous microwave radiation are as follows: the ultrasonic power is 200 W, the ultrasonic frequency is 30 kHz, and it works in a pulsed mode for 20 s with an interval of 10 s; the specific conditions for microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 8 column volumes of eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluate is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0028] Comparative Example 5 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.0 at a liquid-solid ratio of 3:1. Add neutral protease according to a mass ratio of neutral protease to pretreatment product of 0.5:100, oscillate at a rotation speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a rotation speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.5, first add alkaline protease, and perform ultrasonic-assisted synchronous microwave radiation. React at a temperature of 42 °C for 2 h, adjust the pH to 6.5, then add tyrosinase, and control the aeration rate at 0.5 vvm at 25 °C for enzymatic hydrolysis for 3 h to obtain an enzymatic hydrolysate. Among them, the mass ratio of alkaline protease, tyrosinase to pretreatment product is 0.5:0.5:120. The specific conditions of ultrasonic in ultrasonic-assisted synchronous microwave radiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s and intermittent for 10 s; the specific conditions of microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymolysis solution by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect 8 column volumes of eluate and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the ion exchange chromatography conditions are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluate is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0029] Comparative Example 6 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100. Oscillate at a speed of 100 rpm at a temperature of 40 °C for 2 h, centrifuge at a speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, first add neutral protease and perform ultrasonic treatment. React at a temperature of 42 °C for 2 h, adjust the pH to 6.5, then add tyrosinase, and control the ventilation volume to 0.5 vvm at 25 °C and enzymolyze for 3 h to obtain an enzymolysis solution. Among them, the mass ratio of neutral protease, tyrosinase to the pretreatment product is 0.5:0.5:120. The ultrasonic treatment conditions are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, working in pulse mode for 20 s and intermittent for 10 s; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect the eluate with a volume of 8 column volumes and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration factor is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the conditions for ion exchange chromatography are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluate is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0030] Comparative Example 7 An extraction process for mussel protein for skin repair, comprising the following extraction steps: Step 1: Take fresh Mytilus edulis foot gland filaments, rinse them 3 times with physiological saline, and crush them at low temperature to a particle size of 1 mm to obtain a pretreatment product; Step 2: Add the pretreatment product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1. Add alkaline protease according to a mass ratio of alkaline protease to pretreatment product of 0.5:100, and oscillate at a speed of 100 rpm at a temperature of 40 °C for 2 h. Centrifuge at a speed of 8000 rpm for 10 min, and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0, first add neutral protease, perform microwave radiation treatment, react at a temperature of 42 °C for 2 h, adjust the pH to 6.5, then add tyrosinase, and control the aeration rate at 0.5 vvm at 25 °C for 3 h to obtain an enzymatic hydrolysate. Among them, the mass ratio of neutral protease, tyrosinase to pretreatment product is 0.5:0.5:120. The specific conditions for microwave radiation are controlled in segments, with a power of 400 W in the first 15 min and then adjusted to 200 W; Step 4: Remove lipids from the enzymatic hydrolysate by supercritical CO2 extraction, followed by ultrafiltration concentration and ion exchange chromatography. Collect the eluate with a volume of 8 times the column volume and freeze-dry it to obtain mussel protein for skin repair. Among them, the supercritical CO2 extraction pressure is 20 MPa, the extraction temperature is 35 °C, the extraction time is 1 h, and the CO2 flow rate is 5 L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cut-off of 10 kDa, the pressure is 0.1 MPa, the temperature is 4 °C, the pH is 6.5, the concentration multiple is 5 times, and the cross-flow velocity of the ultrafiltration membrane is 1 m / s; the conditions for ion exchange chromatography are as follows: the packing is CM Sepharose FF cation exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4, the eluent is an acetic acid buffer solution with a pH of 4 containing 0.2 mol / L NaCl, the column inner diameter is 1 cm, the column length is 20 cm, the flow rate is 1 mL / min, and the operating temperature is 25 °C.

[0031] Performance Test The comprehensive performance of the mussel protein for skin repair prepared in Examples 1-3 and Comparative Examples 1-7 of this application is as follows: Yield: Yield (%) = mass of purified mussel protein (g) / initial mass of raw material (g) × 100%; Purity: Determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE); Endotoxin content: The Limulus reagent method is used to detect the bacterial endotoxin content in the mussel protein sample; Antioxidant activity: The DPPH radical scavenging rate of mussel protein (concentration of 0.2 mg / mL) is determined by spectrophotometry; The specific test results are shown in Table 1 below and Figure 1 .

[0032] Table 1 Performance parameters of mussel protein prepared in Examples 1-3 and Comparative Examples 1-7

[0033] As can be seen from Table 1, the extraction process provided by this application can efficiently prepare mussel protein, which not only has a high yield, high purity, and low impurity content, but also effectively controls the endotoxin content, thus ensuring the biological safety of the product. In addition, the prepared mussel protein also exhibits excellent antioxidant activity, can effectively scavenge free radicals, and delay the oxidation reaction process, which provides broad application prospects for it in the fields of medicine, cosmetics, etc.

[0034] This specific embodiment is only an explanation of this application and does not limit this application. Those skilled in the art can make modifications without creative contributions to this embodiment according to their needs after reading this specification, but as long as they are within the scope of the claims of this application, they are protected by the patent law.

Claims

1. An extraction process of mussel protein for skin repair, characterized in that, It includes the following extraction steps: Step 1: Take fresh mussel byssus glands, rinse them with physiological saline 3 - 5 times, and crush them at low temperature to a particle size of 1 - 2 mm to obtain a pretreatment product; Step 2: Add the pretreatment product into a citric acid - disodium hydrogen phosphate buffer solution with a pH of 7.5 - 8.0, add alkaline protease, and perform oscillating treatment at a temperature of 40 - 45 °C for 2 - 3 h. Then centrifuge and take the supernatant to obtain supernatant a; Step 3: Adjust the pH of supernatant a to 7.0 - 7.

2. First, add neutral protease, and perform ultrasonic - assisted synchronous microwave radiation. React at a temperature of 42 - 48 °C for 2 - 3 h. Then adjust the pH to 6.5 - 6.8, add tyrosinase, and control the ventilation volume at 25 - 37 °C and enzymatically hydrolyze for 3 - 4 h to obtain an enzymatic hydrolysate; Step 4: Use supercritical CO₂ extraction to remove lipids from the enzymatic hydrolysate, perform ultrafiltration concentration and ion - exchange chromatography technology, collect the eluate with a volume of 8 - 10 times the column volume, and freeze - dry to obtain mussel protein for skin repair.

2. The extraction process of mussel protein for skin repair according to claim 1, characterized in that, In step 2, the mass ratio of alkaline protease to the pretreatment product is 0.5 - 2:100 - 150.

3. The extraction process of mussel protein for skin repair according to claim 1, characterized in that, In step 3, the mass ratio of neutral protease, tyrosinase to the pretreatment product is 0.5 - 1.2:0.5 - 0.8:120 - 150.

4. The extraction process of mussel protein for skin repair according to claim 1, characterized in that, In the ultrasonic - assisted synchronous microwave radiation in step 3, the specific conditions of ultrasound are: ultrasound power is 200 - 400 W, ultrasound frequency is 30 - 40 kHz, pulse mode works for 20 - 30 s, and the interval is 10 - 15 s; the specific conditions of microwave radiation are controlled in segments. The power is 400 - 600 W in the first 15 - 30 min, and then the power is adjusted to 200 - 300 W.

5. The extraction process of mussel protein for skin repair according to claim 1, wherein In step 3, the ventilation volume is 0.5 - 2 vvm.

6. The extraction process of mussel protein for skin repair according to claim 1, characterized in that, In step 4, the supercritical CO₂ extraction pressure is 20 - 25 MPa, the extraction temperature is 35 - 40 °C, the extraction time is 1 - 2 h, and the CO₂ flow rate is 5 - 15 L / min.

7. The extraction process of mussel protein for skin repair according to claim 1, characterized in that, The ultrafiltration concentration conditions in step 4 are: the molecular weight cut - off of the ultrafiltration membrane is 10 - 30 kDa, the pressure is 0.1 - 0.3 MPa, the temperature is 4 - 25 °C, the pH is 6.5 - 7.5, the concentration multiple is 5 - 10 times, and the cross - flow velocity is 1 - 3 m / s.

8. According to the extraction process of mussel protein for skin repair described in claim 7, the ultrafiltration membrane is one of polysulfone ultrafiltration membrane, polyethersulfone ultrafiltration membrane, and polyacrylonitrile ultrafiltration membrane.

9. According to the extraction process of mussel protein for skin repair described in claim 1, the ion - exchange chromatography conditions in step 4 are: the packing is CM Sepharose FF cation - exchange resin, the buffer solution is an acetic acid buffer solution with a pH of 4 - 6, the eluent is an acetic acid buffer solution with a pH of 4 - 6 containing 0 - 1 mol / L NaCl, the inner diameter of the column is 1 - 2 cm, the column length is 20 - 50 cm, the flow rate is 1 - 3 mL / min, and the operating temperature is 25 - 30 °C.

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