A process for extracting mussel protein for skin repair

Abstract

The present application relates to the field of biological extraction technology, and in particular to a kind of extraction process of mussel protein for skin repair, the extraction step of the mussel protein is as follows: step 1, take fresh blue mussel foot silk gland, rinse, low temperature crushing, obtain pre-treated material; step 2, add alkaline protease to the pre-treated material, oscillate, centrifuge, and obtain supernatant a; step 3, adjust supernatant a and first add neutral protease, ultrasound-assisted synchronous microwave radiation, then add tyrosinase, control ventilation, enzymolysis, and obtain enzymolysis solution; step 4, the enzymolysis solution is extracted with supercritical CO2, concentrated by ultrafiltration, ion exchange chromatography technology, eluate is collected, freeze-dried, and mussel protein for skin repair is obtained. The prepared mussel protein not only has high yield, high purity, low impurity content, but also effectively controls endotoxin content, thereby ensuring the biosafety of the product, but also can effectively scavenge free radicals and delay the oxidation reaction process.

Description

Technical Field

[0001] The present application relates to the field of biological extraction technology, and more specifically, to a process for extracting mussel protein for skin repair. Background Art

[0002] Currently, skin repair products on the market rely heavily on chemically synthesized ingredients or plant extracts. Chemically synthesized ingredients are often highly irritating and can cause adverse reactions such as skin allergies and redness. While plant extracts are relatively mild, they have limitations in their repair effects, making them difficult to meet the demand for highly effective skin repair. Therefore, finding a safe and effective skin repair ingredient has become a crucial research direction.

[0003] Mussel protein, a natural marine bioactive protein, has garnered widespread attention in recent years due to its unique structure and functionality. Derived primarily from the foot glands of mussels, its molecular structure is rich in dopa groups (3,4-dihydroxyphenylalanine). Due to its unique biocompatibility, antioxidant properties, and adhesive properties, mussel protein has significant potential in the field of skin repair.

[0004] However, existing mussel protein extraction processes mostly rely on salting out, multiple centrifugation, chromatographic purification and other methods, which are lengthy and require high equipment investment, resulting in a long extraction cycle and low protein recovery rate. High-temperature extraction and strong acid / alkali treatment can easily destroy the dopa group and lysine positive charge structure of mussel mucin, resulting in a significant decrease in cell adhesion and antioxidant functions. Based on the above statements, the present application provides an efficient, environmentally friendly extraction process that retains the dopa group and biological activity of mussel protein for use in the field of skin repair. Summary of the Invention

[0005] In order to solve the technical problems mentioned in the background technology, the present application provides a process for extracting mussel protein for skin repair.

[0006] A process for extracting mussel protein for skin repair adopts the following technical solution:

[0007] Step 1: Take fresh blue mussel foot gland, rinse with normal saline 3-5 times, and grind at low temperature to a particle size of 1-2 mm to obtain a pretreated product;

[0008] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5-8.0 at a liquid-to-solid ratio of 3-5:1, adding alkaline protease, shaking at a speed of 100-200 rpm and a temperature of 40-45° C. for 2-3 hours, centrifuging, and collecting the supernatant to obtain supernatant a;

[0009] Step 3, adjusting the pH of the supernatant a to 7.0-7.2, first adding neutral protease, and subjecting to ultrasound-assisted synchronous microwave irradiation at a temperature of 42-48° C. for 2-3 hours, adjusting the pH to 6.5-6.8, then adding tyrosinase, and incubating at 25-37° C. with controlled ventilation for 3-4 hours to obtain an enzymatic hydrolyzate;

[0010] Step 4: Use supercritical CO2 extraction to remove lipids from the enzymatic hydrolysate, and then use ultrafiltration concentration and ion exchange chromatography technology to collect 8-10 times the column volume of the eluate, and freeze-dry it to obtain the mussel protein for skin repair.

[0011] Preferably, in step 2, the mass ratio of alkaline protease to pretreated material is 0.5-2:100-150.

[0012] Preferably, in step 3, the mass ratio of neutral protease, tyrosinase and pretreated material is 0.5-1.2:0.5-0.8:120-150.

[0013] Preferably, the specific conditions of ultrasound in the ultrasound-assisted synchronous microwave irradiation in step 3 are ultrasonic power 200-400W, ultrasonic frequency 30-40kHz, pulse mode operation 20-30s, and pause 10-15s; the specific conditions of microwave irradiation are segmented control, with the power being 400-600W in the first 15-30min, and then adjusted to 200-300W.

[0014] Preferably, the ventilation volume in step 3 is 0.5-2 vvm.

[0015] Preferably, in step 4, the supercritical CO2 extraction pressure is 20-25 MPa, the extraction temperature is 35-40°C, the extraction time is 1-2h, and the CO2 flow rate is 5-15 L / min.

[0016] Preferably, the ultrafiltration concentration conditions in step 4 are: ultrafiltration membrane cutoff molecular weight of 10-30 kDa, pressure of 0.1-0.3 MPa, temperature of 4-25° C., pH of 6.5-7.5, concentration multiple of 5-10 times, and cross-flow velocity of 1-3 m / s.

[0017] Preferably, the ultrafiltration membrane is one of a polysulfone ultrafiltration membrane, a polyethersulfone ultrafiltration membrane, and a polyacrylonitrile ultrafiltration membrane.

[0018] Preferably, the ion exchange chromatography conditions in step 4 are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4-6, the eluent is an acetate buffer with a pH of 4-6 containing 0-1 mol / L NaCl, the column inner diameter 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.

[0019] In summary, this application has the following beneficial effects:

[0020] In this mussel protein extraction process, the present application sequentially utilizes three enzymes: alkaline protease, neutral protease, and tyrosinase. This process achieves high enzymatic hydrolysis efficiency and a high product yield. During the enzymatic hydrolysis process, alkaline protease specifically hydrolyzes peptide bonds formed by alkaline amino acids, effectively disrupting the complex protein structure of the mussel byssus gland and preliminarily degrading large, impure proteins. Neutral protease then acts on peptide bonds formed by neutral amino acids, further decomposing protein fragments not fully hydrolyzed by the alkaline protease. This expands the enzymatic hydrolysis range and increases the protein's solubility and degree of hydrolysis. Finally, tyrosinase catalyzes the oxidation of tyrosine residues to form DOPA and dopaquinone, key functional groups in mussel byssus protein. Mussel byssus protein, rich in DOPA compounds, exhibits strong adhesion and biocompatibility. It can bind tightly to skin tissue in moist environments, forming hydrogen or covalent bonds with collagen, fibronectin, and other proteins in the skin, enhancing the protein's adhesion and bioactivity. Tyrosinase-catalyzed formation of dopa-like compounds can further participate in intermolecular crosslinking of proteins (such as forming isopeptide bonds or quinone-amino crosslinks), optimizing the protein's spatial structure and imparting enhanced mechanical properties and stability. Furthermore, after tyrosinase treatment, the introduction of charged dopaquinones improves the selectivity and purity of chromatographic separations. Mild enzymatic hydrolysis preserves the functional structure of mussel protein, preventing excessive hydrolysis from damaging its skin-repairing activities, such as promoting collagen synthesis, and its antioxidant and anti-inflammatory properties. The three enzymes work synergistically, targeting different peptide bonds and protein structures through step-by-step adjustments of pH and temperature, enhancing protein extraction efficiency while avoiding the limitations of single enzyme treatment.

[0021] In the mussel protein extraction process, this application utilizes ultrasound-assisted synchronous microwave radiation technology to synergistically enhance the efficiency of enzymatic hydrolysis and improve the protein release rate. The cavitation bubbles generated by high-frequency vibrations can break down the cell structure of the foot silk gland of the blue mussel, accelerate the cell wall / membrane fragmentation, and make the mussel protein in the cell more easily released into the solution, while increasing 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 evenly heat the system, then reduce the power to maintain a constant temperature, avoiding local overheating that causes enzyme inactivation; they can also promote molecular polar motion, accelerate the collision frequency between the substrate and the enzyme, significantly shorten the enzymatic hydrolysis time, and increase the enzymatic hydrolysis reaction rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the SDS-polyacrylamide gel electrophoresis diagram of the mussel protein (3 mg / mL) prepared in Example 1; wherein, lane 1 is the protein marker; lane 2 is the supernatant a with a spotted amount of 3 μL; lane 3 is the enzymatic hydrolyzate with a spotted amount of 3 μL; lane 4 is the mussel protein resolubilized product with a spotted amount of 3 μL. DETAILED DESCRIPTION

[0023] The present application is further described in detail below with reference to the embodiments.

[0024] 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 Yuanzhiyuan Biotechnology Co., Ltd.; tyrosinase (catalog number: GA7589) was purchased from Hubei Guangao Biotechnology Co., Ltd.; CM Sepharose FF cation exchange resin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0025] Examples 1-3 provide a process for extracting mussel protein for skin repair. Example 1

[0026] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0027] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0028] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0029] Step 3, adjusting the pH of the supernatant a to 7.0, first adding neutral protease, ultrasonically assisted synchronous microwave irradiation, reacting at a temperature of 42°C for 2 hours, adjusting the pH to 6.5, then adding tyrosinase, and controlling the ventilation volume at 0.5 vvm at 25°C for 3 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of neutral protease, tyrosinase and pretreated material is 0.5:0.5:120, and the specific ultrasonic conditions in the ultrasonic-assisted synchronous microwave irradiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, pulse mode operation for 20 seconds, and rest for 10 seconds; the specific microwave irradiation conditions are segmented control, the power is 400 W in the first 15 minutes, and the power is adjusted to 200 W thereafter;

[0030] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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

[0031] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0032] Step 1: fresh blue mussel foot gland was taken, rinsed with normal saline 4 times, and crushed at low temperature to a particle size of 1.5 mm to obtain a pretreated product;

[0033] Step 2: The pretreated material was added to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.8 at a liquid-to-solid ratio of 4:1, and alkaline protease was added at a mass ratio of alkaline protease to pretreated material of 1:125. The mixture was shaken at a speed of 150 rpm and a temperature of 42°C for 2.5 hours, and centrifuged at a speed of 9000 rpm for 12 minutes. The supernatant was collected to obtain supernatant a.

[0034] Step 3, adjusting the pH of the supernatant a to 7.1, first adding neutral protease, ultrasonically assisted synchronous microwave irradiation, reacting at a temperature of 45°C for 2.5 hours, adjusting the pH to 6.6, then adding tyrosinase, controlling the ventilation volume at 1 vvm at 32°C, and enzymolyzing for 3.5 hours to obtain an enzymatic solution, wherein the mass ratio of neutral protease, tyrosinase and pretreated material is 0.8:0.6:135, and the specific ultrasonic conditions in the ultrasonic-assisted synchronous microwave irradiation are ultrasonic power 300 W, ultrasonic frequency 35 kHz, pulse mode operation for 25 seconds, and intermission of 12 seconds; the specific microwave irradiation conditions are segmented control, with a power of 500 W in the first 20 minutes and then adjusted to 250 W;

[0035] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 9 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein the supercritical CO2 extraction pressure is 22MPa, the extraction temperature is 38°C, the extraction time is 1.5h, and the CO2 flow rate is 10L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane, the ultrafiltration membrane has a molecular weight cutoff of 20kDa, the pressure is 0.2MPa, the temperature is 20°C, the pH is 7.0, the concentration multiple is 8 times, and the ultrafiltration membrane cross flow velocity is 2m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 5, the eluent is an acetate buffer with a pH of 5 containing 0.5mol / LNaCl, the column inner diameter is 1.5cm, the column length is 35cm, the flow rate is 2mL / min, and the operating temperature is 28°C. Example 3

[0036] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0037] Step 1: Take fresh blue mussel foot gland, rinse with normal saline 5 times, and grind at low temperature to a particle size of 2 mm to obtain a pretreated product;

[0038] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 8.0 at a liquid-solid ratio of 5:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 2:150, shaking at a speed of 200 rpm and a temperature of 45°C for 3 hours, centrifuging at a speed of 10,000 rpm for 15 minutes, and taking the supernatant to obtain supernatant a;

[0039] Step 3, adjusting the pH of the supernatant a to 7.2, first adding neutral protease, ultrasonically assisted synchronous microwave irradiation, reacting at a temperature of 48°C for 3 hours, adjusting the pH to 6.8, then adding tyrosinase, controlling the ventilation volume at 2vvm at 37°C, and performing enzymatic hydrolysis for 4 hours to obtain an enzymatic solution, wherein the mass ratio of neutral protease, tyrosinase and pretreated material is 1.2:0.8:150, and the specific ultrasonic conditions in the ultrasonic-assisted synchronous microwave irradiation are ultrasonic power 400W, ultrasonic frequency 40kHz, pulse mode operation for 30s, and intermission of 15s; the specific microwave irradiation conditions are segmented control, with a power of 600W in the first 30 minutes and then adjusted to 300W;

[0040] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 10 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein the supercritical CO2 extraction pressure is 25MPa, the extraction temperature is 40°C, the extraction time is 2h, and the CO2 flow rate is 15L / min; the ultrafiltration membrane is a polysulfone ultrafiltration membrane, the ultrafiltration membrane has a molecular weight cutoff of 30kDa, the pressure is 0.3MPa, the temperature is 25°C, the pH is 7.5, the concentration multiple is 10 times, and the ultrafiltration membrane cross flow velocity is 3m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 6, the eluent is an acetate buffer with a pH of 6 containing 1.0mol / LNaCl, the column inner diameter is 2cm, the column length is 50cm, the flow rate is 3mL / min, and the operating temperature is 30°C.

[0041] Comparative Example 1

[0042] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0043] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0044] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-to-solid ratio of 3:1, shaking at a speed of 100 rpm and a temperature of 40° C. for 2 h, centrifuging at a speed of 8000 rpm for 10 min, and collecting the supernatant to obtain supernatant a;

[0045] Step 3, adjusting the pH of the supernatant a to 7.0, first adding neutral protease, ultrasonic-assisted synchronous microwave irradiation, reacting at a temperature of 42°C for 2h, adjusting the pH to 6.5, then adding tyrosinase, controlling the ventilation volume at 0.5vvm at 25°C, and enzymolysis for 3h to obtain an enzymatic solution, wherein the mass ratio of neutral protease, tyrosinase and pretreated material is 0.5:0.5:120. The specific ultrasonic conditions in the ultrasonic-assisted synchronous microwave irradiation are ultrasonic power of 200W, ultrasonic frequency of 30kHz, pulse mode operation for 20s, and rest for 10s; the specific microwave irradiation conditions are segmented control, the power is 400W in the first 15min, and then the power is adjusted to 200W;

[0046] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0047] Comparative Example 2

[0048] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0049] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0050] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0051] Step 3, adjusting the pH of the supernatant a to 7.0, performing ultrasound-assisted synchronous microwave irradiation, reacting at a temperature of 42°C for 2 hours, adjusting the pH to 6.5, adding tyrosinase, controlling the ventilation volume to 0.5 vvm at 25°C, and performing enzymatic hydrolysis for 3 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of tyrosinase to the pretreated material is 1:120, and the specific ultrasound conditions in the ultrasound-assisted synchronous microwave irradiation are an ultrasonic power of 200 W, an ultrasonic frequency of 30 kHz, a pulse mode of operation for 20 seconds, and an interval of 10 seconds; the specific microwave irradiation conditions are segmented control, with a power of 400 W in the first 15 minutes, and then the power is adjusted to 200 W;

[0052] Step 4: The enzymatic hydrolysate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0053] Comparative Example 3

[0054] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0055] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0056] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0057] Step 3, adjusting the pH of the supernatant a to 7.0, adding neutral protease, and subjecting to ultrasound-assisted synchronous microwave irradiation. The reaction was carried out at a temperature of 42° C. for 5 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of neutral protease to pretreated material was 1:120, and the specific ultrasonic conditions in the ultrasound-assisted synchronous microwave irradiation were as follows: ultrasonic power 200 W, ultrasonic frequency 30 kHz, pulse mode operation for 20 seconds, and rest for 10 seconds; the specific microwave irradiation conditions were segmented control, with a power of 400 W in the first 15 minutes, and then the power was adjusted to 200 W;

[0058] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0059] Comparative Example 4

[0060] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0061] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0062] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0063] Step 3, adjusting the pH of the supernatant a to 7.0, adding neutral protease and tyrosinase, and subjecting to ultrasound-assisted synchronous microwave irradiation. At a temperature of 42° C., controlling the ventilation volume to 0.5 vvm, and performing enzymatic hydrolysis for 5 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of neutral protease, tyrosinase, and pretreated material is 0.5:0.5:120, and the specific ultrasonic conditions in the ultrasound-assisted synchronous microwave irradiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, pulse mode operation for 20 seconds, and rest for 10 seconds; the specific microwave irradiation conditions are segmented control, with a power of 400 W in the first 15 minutes, and then the power is adjusted to 200 W;

[0064] Step 4: The enzymatic hydrolysate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0065] Comparative Example 5

[0066] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0067] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0068] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.0 at a liquid-solid ratio of 3:1, adding neutral protease at a mass ratio of neutral protease to pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0069] Step 3, adjusting the pH of the supernatant a to 7.5, first adding alkaline protease, and subjecting to ultrasound-assisted synchronous microwave irradiation, reacting at a temperature of 42°C for 2 hours, adjusting the pH to 6.5, and then adding tyrosinase. At 25°C, the ventilation volume is controlled to 0.5 vvm, and enzymolysis is carried out for 3 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of alkaline protease, tyrosinase and pretreated material is 0.5:0.5:120, and the specific ultrasonic conditions in the ultrasound-assisted synchronous microwave irradiation are ultrasonic power of 200 W, ultrasonic frequency of 30 kHz, pulse mode operation for 20 seconds, and rest for 10 seconds; the specific microwave irradiation conditions are segmented control, the power is 400 W in the first 15 minutes, and the power is adjusted to 200 W thereafter;

[0070] Step 4: The enzymatic hydrolyzate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0071] Comparative Example 6

[0072] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0073] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0074] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0075] Step 3, adjusting the pH of the supernatant a to 7.0, first adding neutral protease, ultrasonically treating, reacting at a temperature of 42°C for 2 hours, adjusting the pH to 6.5, then adding tyrosinase, controlling the ventilation volume at 0.5 vvm at 25°C, and enzymolyzing for 3 hours to obtain an enzymatic hydrolyzate, wherein the mass ratio of neutral protease, tyrosinase and pretreated material is 0.5:0.5:120, and the ultrasonic treatment conditions are ultrasonic power 200 W, ultrasonic frequency 30 kHz, pulse mode operation for 20 seconds, and rest for 10 seconds;

[0076] Step 4: The enzymatic hydrolysate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0077] Comparative Example 7

[0078] A process for extracting mussel protein for skin repair, comprising the following extraction steps:

[0079] Step 1: Take fresh blue mussel foot gland, rinse with normal saline three times, and grind at low temperature to a particle size of 1 mm to obtain a pretreated product;

[0080] Step 2: adding the pretreated material to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5 at a liquid-solid ratio of 3:1, adding alkaline protease at a mass ratio of alkaline protease to the pretreated material of 0.5:100, shaking at a speed of 100 rpm and a temperature of 40°C for 2 hours, centrifuging at a speed of 8000 rpm for 10 minutes, and taking the supernatant to obtain supernatant a;

[0081] Step 3: adjusting the pH of the supernatant a to 7.0, first adding neutral protease, and subjecting to microwave radiation treatment at a temperature of 42° C. for 2 h, adjusting the pH to 6.5, and then adding tyrosinase. At 25° C., controlling the ventilation volume to 0.5 vvm, and enzymolysis for 3 h to obtain an enzymatic hydrolyzate, wherein the mass ratio of neutral protease, tyrosinase, and pretreated material is 0.5:0.5:120, and the specific conditions of microwave radiation are segmented control, with a power of 400 W in the first 15 min and then adjusted to 200 W;

[0082] Step 4: The enzymatic hydrolysate is extracted with supercritical CO2 to remove lipids, and the eluate is collected by ultrafiltration concentration and ion exchange chromatography technology, and 8 times the column volume is freeze-dried to obtain mussel protein for skin repair, wherein 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, the ultrafiltration membrane has a molecular weight cutoff 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 ultrafiltration membrane cross-flow velocity is 1 m / s; the ion exchange chromatography conditions are: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4, the eluent is an acetate buffer 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.

[0083] Performance Testing

[0084] The comprehensive properties of the mussel proteins for skin repair prepared in Examples 1-3 and Comparative Examples 1-7 of the present application are as follows:

[0085] Yield: Yield (%) = mass of purified mussel protein (g) / initial mass of raw material (g) × 100%;

[0086] Purity: Determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE);

[0087] Endotoxin content: The bacterial endotoxin content in mussel protein samples was detected using the Limulus amebocyte lysate (LAL) assay.

[0088] Antioxidant activity: The DPPH radical scavenging rate of mussel protein (concentration 0.2 mg / mL) was determined by spectrophotometry;

[0089] The specific test results are shown in Table 1 and Figure 1 .

[0090] Table 1 Performance parameters of mussel proteins prepared in Examples 1-3 and Comparative Examples 1-7

[0091]

[0092] As can be seen from Table 1, the extraction process provided in this application can efficiently prepare mussel protein, which not only has high yield, high purity, and low impurity content, but also effectively controls the endotoxin content, thereby ensuring the biosafety 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 it with broad application prospects in the fields of medicine, cosmetics, etc.

[0093] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A process for extracting mussel protein for skin repair, characterized in that: The extraction steps include: Step 1: Take fresh blue mussel foot gland, rinse with normal saline 3-5 times, and grind at low temperature to a particle size of 1-2 mm to obtain a pretreated product; Step 2: adding the pretreated product to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 7.5-8.0, adding alkaline protease, shaking the mixture at a temperature of 40-45° C. for 2-3 hours, centrifuging, and collecting the supernatant to obtain supernatant a; Step 3, adjusting the pH of the supernatant a to 7.0-7.2, first adding neutral protease, and subjecting to ultrasound-assisted synchronous microwave irradiation at a temperature of 42-48° C. for 2-3 hours, adjusting the pH to 6.5-6.8, then adding tyrosinase, and incubating at 25-37° C. with controlled ventilation for 3-4 hours to obtain an enzymatic hydrolyzate; Step 4: extract the enzymatic hydrolysate with supercritical CO2 to remove lipids, concentrate through ultrafiltration, and use ion exchange chromatography technology to collect 8-10 column volumes of the eluate, and freeze-dry to obtain mussel protein for skin repair; In step 2, the mass ratio of alkaline protease to pretreated material is 0.5-2:100-150; In step 3, the mass ratio of neutral protease, tyrosinase and pretreated material is 0.5-1.2:0.5-0.8:120-150; In the ultrasound-assisted synchronous microwave irradiation in step 3, the specific ultrasonic conditions are as follows: ultrasonic power 200-400W, ultrasonic frequency 30-40kHz, pulse mode operation 20-30s, rest 10-15s; the specific microwave irradiation conditions are segmented control, the power is 400-600W in the first 15-30 minutes, and then the power is adjusted to 200-300W; The ion exchange chromatography conditions in step 4 are as follows: the filler is CM Sepharose FF cation exchange resin, the buffer is an acetate buffer with a pH of 4-6, the eluent is an acetate buffer with a pH of 4-6 containing 0-1 mol / L NaCl, the column inner diameter 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.

2. The process for extracting mussel protein for skin repair according to claim 1, characterized in that: The ventilation volume in step 3 is 0.5-2 vvm.

3. 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 hours, and the CO2 flow rate is 5-15 L / min.

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

5. The process for extracting mussel protein for skin repair according to claim 4, wherein the ultrafiltration membrane is one of a polysulfone ultrafiltration membrane, a polyethersulfone ultrafiltration membrane, and a polyacrylonitrile ultrafiltration membrane.

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