Preparation method of undaria pinnatifida protein with antioxidant activity

Abstract

The invention discloses a preparation method of undaria pinnatifida protein with antioxidant activity, which comprises the following steps: firstly, effectively destroying the cell wall structure of undaria pinnatifida by using a compound enzyme to fully release protein in cells, and further obtaining undaria pinnatifida crude protein by using acid-base treatment and an isoelectric precipitation method; and separating and purifying the extracted crude protein through a PEG / (NH4) 2SO4 aqueous two-phase system. According to the method, a polyethylene glycol (PEG) / (NH4) 2SO4 aqueous two-phase system is adopted, so that polyphenol, pigment and other impurities in the undaria pinnatifida protein can be effectively removed, and the purity of the undaria pinnatifida protein is effectively improved; and step-by-step acid and alkali treatment is adopted, so that the influence of algin precipitation on the protein can be avoided, the extraction rate of the protein is effectively improved, the undaria pinnatifida protein with high purity and strong antioxidant activity is further obtained, and the undaria pinnatifida protein has relatively high commercial value. Meanwhile, the method has the advantages of simple steps, easiness in operation and the like, and is suitable for industrial large-scale production.

Description

Technical Field

[0001] The invention relates to a method for preparing undaria protein, in particular to a method for preparing undaria protein with antioxidant activity. Background Art

[0002] Brown algae has a very low fat content, but contains a large amount of polysaccharides and rich proteins and trace elements, among which the crude protein content is 8-20%, such as the crude protein content of kelp is about 18%. A large number of studies have shown that brown algae has a variety of biological functions such as lowering blood lipids, lowering blood sugar, regulating immunity, anti-coagulation, anti-tumor, lead detoxification and anti-oxidation, and has attracted widespread attention. However, due to the large amount of sticky polysaccharides in brown algae tissues, which hinders the dissolution of protein, the efficiency of extracting kelp protein by existing methods is very low, which affects the further research and utilization of brown algae protein. Therefore, the current research on brown algae mainly focuses on components such as alginate, fucoidan sulfate, brown algae polyphenols, fucoxanthin and dietary fiber. The development of effective extraction and separation technology of protein in brown algae resources is of great significance to the development and utilization of algae protein resources in the later stage. Summary of the invention

[0003] The present invention aims to solve the above technical problems existing in the prior art and provides a method for preparing kelp protein with antioxidant activity.

[0004] The technical solution of the present invention is: a method for preparing kelp protein with antioxidant activity, which is carried out in the following steps: Step 1. Wash and drain the kelp, and then perform high-speed homogenization and wall-breaking treatment to obtain a kelp homogenate; Step 2. Weigh the kelp homogenate, add distilled water according to the material-liquid mass volume ratio of 1g:60-100mL, add 0.1%-0.6% cellulase, 0.02%-0.14% pectinase, and 0.04-0.2% β-glucosidase by the mass of the raw materials, control the enzymatic hydrolysis pH at 4.0-6.5, the enzymatic hydrolysis temperature at 40-60°C, and after enzymatic hydrolysis for 0.5-2.5 h, inactivate the enzyme at 85°C for 10 min to obtain an enzymatic solution, wherein the enzymatic activities of the cellulase and pectinase are both 50,000 U / g, and the enzymatic activity of the β-glucosidase is 300 U / g; Step 3. The pH value of the enzymatic hydrolyzate is adjusted to 10-14 with NaOH for alkali extraction at a temperature of 50-70°C for 1-3 hours to obtain a kelp protein extract; Step 4. The pH value of the kelp protein extract was adjusted to 2.5 with 1 mol / L HCl, and then adjusted to 3.7 with NaOH. After standing for 1 hour, the extract was centrifuged at 10,000 r / min for 10-20 min, and the precipitate was the kelp crude protein. Step 5. Mix PEG2000 and (NH4)2SO4 to prepare a mixed solution, and then mix it with kelp crude protein, wherein the mass percentage of PEG2000 in the mixed solution is 10-30%, the mass percentage of (NH4)2SO4 in the mixed solution is 12-20%, and the dosage ratio of kelp crude protein to the mixed solution is 4.0 mg / g; adjust the pH value to 4-8, vortex mix, let stand for 3 hours, centrifuge at 4000 r / min for 10 min, take the supernatant and perform 3000Da ultrafiltration, and take the ultrafiltration retentate and concentrate and freeze-dry to obtain kelp protein.

[0005] Preferably, the kelp is salted kelp, which is first washed to remove salt particles attached to its surface, and then soaked for 1 hour, and then washed and soaked again for 3 times. After the raw material is desalted and drained, it is subjected to high-speed homogenization and wall breaking treatment.

[0006] Preferably, the high-speed homogenization and wall-breaking treatment in step 1 is carried out at 10,000 r / min for 6 min.

[0007] The present invention firstly uses a composite enzyme to effectively destroy the cell wall structure of kelp, so that the protein in the cell is fully released, and then uses acid-base treatment and isoelectric precipitation method to obtain kelp crude protein, and then separates and purifies the extracted crude protein through a (PEG) / (NH4)2SO4 two-phase aqueous system. The polyethylene glycol (PEG) / (NH4)2SO4 two-phase aqueous system used in the present invention can effectively remove impurities such as polyphenols and pigments in kelp protein, and effectively improve the purity of kelp protein; the step-by-step acid-base treatment used can avoid the influence of alginate precipitation on protein, effectively improve the extraction rate of protein, and then obtain kelp protein with high purity and strong antioxidant activity, which has high commercial value. At the same time, the present invention has the advantages of simple steps and easy operation, and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a schematic diagram of the scavenging ability of the kelp protein obtained in Example 2 of the present invention on hydroxyl radicals at different concentrations.

[0009] Figure 2 It is a schematic diagram of the scavenging ability of the kelp protein obtained in Example 2 of the present invention on DPPH free radicals at different concentrations.

[0010] Figure 3 The results of the ABTS assay performed by the kelp protein obtained in Example 2 of the present invention at different concentrations + •Schematic diagram of free radical scavenging ability. DETAILED DESCRIPTION Example 1

[0011] The method for preparing the kelp protein with antioxidant activity of the present invention is carried out in the following steps: Step 1. Wash and drain fresh kelp, and then perform high-speed homogenization and wall-breaking treatment to obtain kelp homogenate; Step 2. Weigh the kelp homogenate, add distilled water at a material-liquid mass volume ratio of 1g:100mL, add 0.1% cellulase, 0.02% pectinase, and 0.1% β-glucosidase by weight of the raw materials in sequence, control the enzymatic hydrolysis pH to 5.0, the enzymatic hydrolysis temperature to 50°C, and after enzymatic hydrolysis for 2.5 hours, inactivate the enzyme at 85°C for 10 minutes to obtain an enzymatic hydrolyzate, wherein the enzymatic activities of the cellulase and pectinase are 50,000U / g, and the enzymatic activity of the β-glucosidase is 300U / g; Step 3. The enzymatic hydrolyzate was adjusted to pH 11 with NaOH for alkali extraction at 60°C for 3 hours to obtain a kelp protein extract; Step 4. In order to prevent alginate precipitation, the pH of the kelp protein extract was first adjusted to 2.5 with 1 mol / L HCl, and then adjusted to 3.7 with NaOH. After standing for 1 hour, the extract was centrifuged at 10,000 r / min for 20 minutes, and the precipitate was the kelp crude protein. Step 5. Mix PEG2000 and (NH4)2SO4 to prepare a mixed solution, and then mix it with kelp crude protein, wherein the mass percentage of PEG2000 in the mixed solution is 10%, the mass percentage of (NH4)2SO4 in the mixed solution is 16%, and the dosage ratio of the kelp crude protein to the mixed solution is 4.0 mg / g; adjust the pH value to 6.5, vortex mix, let stand for 3 hours, centrifuge at 4000 r / min for 10 min, take the supernatant and perform 3000Da ultrafiltration, and take the ultrafiltration retentate and concentrate and freeze-dry to obtain kelp protein.

[0012] The content of water-soluble protein in the extract was determined by the Coomassie brilliant blue method, and the extraction rate of kelp protein was calculated to be 40.01%. The crude protein of kelp was separated and purified by polyethylene glycol (PEG) / (NH4)2SO4 two-phase aqueous system, and the protein extraction efficiency reached 68.88%, that is, 68.88% of the protein was transferred to the upper aqueous phase. Example 2

[0013] The method for preparing the kelp protein with antioxidant activity of the present invention is carried out in the following steps: Step 1. Take salted kelp, wash off the salt particles attached to its surface, soak it for 1 hour, and then wash and soak it again for 3 times to complete the desalination of the raw material; after draining the water, subject the kelp to high-speed homogenization and wall breaking treatment (10000 r / min, 6 min) to obtain kelp homogenate; Step 2. Weigh the kelp homogenate, add distilled water at a material-liquid mass volume ratio of 1g:60-100mL, add 0.1% cellulase, 0.1% pectinase, and 0.06% β-glucosidase by weight of the raw materials in sequence, control the enzymatic hydrolysis pH to 6.0, the enzymatic hydrolysis temperature to 55°C, after enzymatic hydrolysis for 1 hour, inactivate the enzyme at 85°C for 10 minutes to obtain an enzymatic hydrolyzate, wherein the enzymatic activities of the cellulase and pectinase are 50000U / g, and the enzymatic activity of the β-glucosidase is 300U / g; Step 3. The pH value of the enzymatic hydrolyzate was adjusted to 13 with NaOH for alkali extraction at 65°C for 2.5 hours to obtain a kelp protein extract; Step 4. In order to prevent alginate precipitation, the pH of the kelp protein extract was first adjusted to 2.5 with 1 mol / L HCl, and then adjusted to 3.7 with NaOH. After standing for 1 hour, the extract was centrifuged at 10000 r / min for 10 minutes, and the precipitate was the kelp crude protein. Step 5. Mix PEG2000 and (NH4)2SO4 to prepare a mixed solution, and then mix it with kelp crude protein, wherein the mass percentage of PEG2000 in the mixed solution is 25%, the mass percentage of (NH4)2SO4 in the mixed solution is 12%, and the dosage ratio of kelp crude protein to the mixed solution is 4.0 mg / g; adjust the pH value to 6.5, vortex mix, let stand for 3 hours, centrifuge at 4000 r / min for 10 min, take the supernatant and perform 3000Da ultrafiltration, and take the ultrafiltration retentate and concentrate and freeze-dry to obtain kelp protein.

[0014] The content of water-soluble protein in the extract was determined by the Coomassie Brilliant Blue method, and the extraction rate of kelp protein was calculated to be 82.39%. The crude kelp protein was separated and purified by polyethylene glycol (PEG) / (NH4)2SO4 aqueous two-phase system, and the protein extraction efficiency could reach 98.87%. The purity of the obtained protein was higher than 90%, and the polyphenol content was less than 0.8%. Example 3

[0015] The method for preparing the kelp protein with antioxidant activity of the present invention is carried out in the following steps: Step 1. Take salted kelp, wash off the salt particles attached to its surface, soak it for 1 hour, and then wash and soak it again for 3 times to complete the desalination of the raw material; after draining the water, subject the kelp to high-speed homogenization and wall breaking treatment (10000 r / min, 6 min) to obtain kelp homogenate; Step 2. Weigh the kelp homogenate, add distilled water at a material-liquid mass volume ratio of 1g:100mL, add 0.1% cellulase, 0.1% pectinase, and 0.06% β-glucosidase by weight of the raw materials in sequence, control the enzymatic hydrolysis pH to 6.0, the enzymatic hydrolysis temperature to 60°C, and after enzymatic hydrolysis for 2.5 hours, inactivate the enzyme at 85°C for 10 minutes to obtain an enzymatic hydrolyzate, wherein the enzymatic activities of the cellulase and pectinase are 50,000U / g, and the enzymatic activity of the β-glucosidase is 300U / g; Step 3. The pH value of the enzymatic hydrolyzate was adjusted to 14 with NaOH for alkali extraction at a temperature of 50°C for 3 hours to obtain a kelp protein extract; Step 4. In order to prevent alginate precipitation, the pH of the kelp protein extract was first adjusted to 2.5 with 1 mol / L HCl, and then adjusted to 3.7 with NaOH. After standing for 1 hour, the extract was centrifuged at 10,000 r / min for 20 minutes, and the precipitate was the kelp crude protein. Step 5. Mix PEG2000 and (NH4)2SO4 to prepare a mixed solution, and then mix it with kelp crude protein, wherein the mass percentage of PEG2000 in the mixed solution is 25%, the mass percentage of (NH4)2SO4 in the mixed solution is 20%, and the dosage ratio of the kelp crude protein to the mixed solution is 4.0 mg / g; adjust the pH value to 6.5, vortex mix, let stand for 3 hours, centrifuge at 4000 r / min for 10 min, take the supernatant and perform 3000Da ultrafiltration, and take the ultrafiltration retentate and concentrate and freeze-dry to obtain kelp protein.

[0016] The content of water-soluble protein in the extract was determined by the Coomassie Brilliant Blue method, and the extraction rate of kelp protein was calculated to be 66.68%. The crude kelp protein was separated and purified by polyethylene glycol (PEG) / (NH4)2SO4 aqueous two-phase system, and the protein extraction efficiency could reach 68.72%.

[0017] experiment: The kelp protein obtained in Example 2 was subjected to in vitro antioxidant activity assay: The scavenging activity of the protein sample on hydroxyl radicals, DPPH radicals, ABTS+· and hydroxyl radicals was determined by in vitro tests to evaluate its antioxidant activity. The schematic diagrams of the scavenging abilities are shown in Figure 2. Figure 1 , 2 , as shown in Figure 3.

[0018] Depend on Figure 1It can be seen that in the range of 0.8~10.0 mg / mL, kelp protein showed a certain scavenging activity against hydroxyl free radicals. With the increase of protein sample concentration, the scavenging rate of hydroxyl free radicals gradually increased. When the protein sample concentration was 8 mg / mL, the scavenging rate of kelp crude protein against hydroxyl free radicals was close to 100%, IC 50 The value is 1.48 mg / mL.

[0019] Depend on Figure 2 It can be seen that in the range of 0.5~10.0 mg / mL, kelp protein showed a certain scavenging activity against DPPH free radicals, and the concentration level was in a dose-response relationship, that is, with the increase of concentration, the scavenging rate of DPPH by kelp protein samples gradually increased. 50 It was 0.436 mg / mL, indicating that salted kelp protein had good DPPH free radical scavenging ability.

[0020] Depend on Figure 3 It can be seen that within the range of 0.5~10.0 mg / mL, kelp protein has a certain scavenging activity for ABTS+•. As the concentration of the protein sample increases, the scavenging rate of ABTS+• gradually increases, and then tends to be flat. When the concentration is 4 mg / mL, the scavenging rate of kelp protein for ABTS+• reaches 98%, IC 50 The value is 0.574 mg / mL.

[0021] The results show that the extraction rate and purity of the kelp protein prepared by the preparation method of the present invention are high, and the prepared kelp protein has high antioxidant activity.

Claims

1. A method for preparing kelp protein with antioxidant activity, characterized in that Follow the steps below: Step 1. Wash and drain the kelp, and then perform high-speed homogenization and wall-breaking treatment to obtain a kelp homogenate; Step 2. Weigh the kelp homogenate, add distilled water at a material-liquid mass volume ratio of 1g:60-100mL, add 0.1%-0.6% cellulase, 0.02%-0.14% pectinase, and 0.04-0.2% β-glucosidase by weight of the raw materials, control the enzymatic hydrolysis pH to 4.0-6.5, the enzymatic hydrolysis temperature to 40-60°C, and after enzymatic hydrolysis for 0.5-2.5 h, inactivate the enzyme at 85°C for 10 min to obtain an enzymatic hydrolyzate, wherein the enzymatic activities of the cellulase and pectinase are both 50,000U / g, and the enzymatic activity of the β-glucosidase is 300U / g; Step 3. The pH value of the enzymatic hydrolyzate is adjusted to 10-14 with NaOH for alkali extraction at a temperature of 50-70°C for 1-3 hours to obtain a kelp protein extract; Step 4. The pH value of the kelp protein extract was adjusted to 2.5 with 1 mol / L HCl, and then adjusted to 3.7 with NaOH. After standing for 1 hour, the extract was centrifuged at 10,000 r / min for 10-20 min, and the precipitate was the kelp crude protein. Step 5. Mix PEG2000 and (NH4)2SO4 to prepare a mixed solution, and then mix it with kelp crude protein, wherein the mass percentage of PEG2000 in the mixed solution is 10-30%, the mass percentage of (NH4)2SO4 in the mixed solution is 12-20%, and the dosage ratio of kelp crude protein to the mixed solution is 4.0 mg / g; adjust the pH value to 4-8, vortex mix, let stand for 3 hours, centrifuge at 4000 r / min for 10 min, take the supernatant and perform 3000Da ultrafiltration, and take the ultrafiltration retentate and concentrate and freeze-dry to obtain kelp protein.

2. The method for preparing the undaria pinnatifida protein with antioxidant activity according to claim 1, characterized in that: The kelp is salted kelp, and the salt particles attached to the surface are washed off and soaked for 1 hour. After that, it is washed and soaked again for 3 times, and after the raw material is desalted and the water is drained, a high-speed homogenization and wall breaking treatment is performed.

3. The method for preparing the undaria pinnatifida protein with antioxidant activity according to claim 1 or 2, characterized in that: The high-speed homogenization and wall-breaking treatment in step 1 is performed at 10,000 r / min for 6 min.

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