Potato protein with whitening and anti-aging effects as well as extraction and purification method and application thereof

Abstract

The invention discloses a potato protein with whitening and anti-aging effects as well as an extraction and purification method and application thereof, and aims to provide a potato protein capable of remarkably reducing the melanin content in cells, effectively removing various forms of free radicals in the cells and improving the whitening and anti-aging effects. The invention relates to a potato protein capable of protecting cells from oxidative stress damage, an extraction and purification method of the protein and application of the protein as an active additive component of cosmetics. According to the technical scheme, the method comprises the steps that potato total protein is extracted through precipitation, centrifugation, filtration, dialysis, freeze drying and the like, purified protein components are obtained through gel chromatography column separation, and the molecular weight of the protein is 39 kD and 17 kD; relates to the technical field of biology.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a potato protein component with whitening and anti-aging effects, an extraction and purification method thereof, and an application thereof. Background Art

[0002] Potatoes are perennial tuberous herbaceous plants of the Solanaceae family. They are rich in nutrients and are known as "underground apples" and "second bread". They are the fourth largest food crop after wheat, rice and corn. Potatoes contain sugars, vitamins, minerals and proteins. Among them, the protein content of potatoes is about 2%, which is composed of 19 kinds of amino acids. Among them, the content of essential amino acids is high, accounting for 20.1% of the total protein. The proportion of allergenic proteins is low, and it is easily digested and absorbed by the human body, making its nutritional value equivalent to that of eggs, and it is a high-protein crop.

[0003] The protein contained in potatoes can be divided into three parts according to molecular weight: high molecular weight protein, accounting for about 10% of the total protein, with a molecular weight greater than 40kD; glycoprotein, accounting for about 40% of the total protein, with a molecular weight of 39-45kD; protease inhibitors, accounting for about 50% of the total protein, with a molecular weight of 5-25kD.

[0004] Potato protein has many advantages, which makes it have a wide range of application potential in the food industry and nutrition field. Potato protein contains a large amount of essential amino acids, has good solubility, emulsification and gelation, and can be used in food as a high-quality protein source through modification. In addition, some protein components in potatoes also have a variety of biological activities, such as glycoproteins showing high antioxidant activity, potato carboxypeptidase inhibitors, showing anti-cell proliferation activity of various human tumor cells, ACE inhibitory peptides produced after potato protein hydrolysis have a hypotensive effect, and α-lipoic acid contained in potato protein can help the human body convert glucose into energy, help control blood sugar levels, and improve vasodilation function. At the same time, potato extracts can also reduce weight gain and fat accumulation, improve glucose control, and have a positive effect on weight loss and control of diet-induced obesity. Therefore, potato protein has great development value and has been a hot topic of research in the food field in recent years.

[0005] However, the research on potato protein is still in its early stages, mainly focusing on the properties and performance of total potato protein or partial protein mixtures. There is still a lack of systematic research on the purification, properties and efficacy of each protein component. Clarifying the properties and functions of each potato protein component is of great significance for the research and application of potato protein.

[0006] The present invention obtains two purified potato protein components with different molecular weights by optimizing the extraction and purification method, and systematically studies their whitening and anti-aging effects. The experimental results prove that these two potato protein components are expected to become new active ingredients for whitening and anti-aging skin care products. Summary of the invention

[0007] In view of the above problems, the first object of the present invention is to provide potato protein which can significantly reduce the melanin content in cells, effectively remove various forms of free radicals in cells, and protect cells from oxidative stress.

[0008] The second object of the present invention is to provide a method for extracting and purifying the above-mentioned potato protein, which can effectively extract and purify high-purity protein components from potatoes.

[0009] The third object of the present invention is to provide the application of the potato protein as an active additive in cosmetics.

[0010] To this end, the first technical solution provided by the present invention is as follows:

[0011] The potato protein has whitening and anti-aging effects, and the molecular weight of the potato protein is 39kD and 17kD.

[0012] The second technical solution provided by the present invention is a method for extracting and purifying potato protein with whitening and anti-aging effects as described in the first technical solution, which comprises the following steps in sequence:

[0013] 1) washing, peeling and cutting fresh potatoes, grinding them with water, and then adding polyphenol antioxidants to obtain a mixture;

[0014] 2) standing the mixture prepared in step 1) at 3-5° C. to allow starch to precipitate, collecting the supernatant, centrifuging, collecting the supernatant again, and filtering to obtain transparent potato juice;

[0015] 3) dialyzing the potato juice prepared in step 2) at 3-5° C. for 2-4 days, freeze-drying to obtain total potato protein, and storing it in a refrigerator at -20° C. for later use;

[0016] 4) The potato total protein is separated and purified by gel chromatography column, pre-balanced with buffer, the eluate is collected, and the eluate in each tube is freeze-dried to obtain a purified protein component.

[0017] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the mass ratio of potatoes to water in step 1) is 1:1.

[0018] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the polyphenol antioxidant described in step 1) is a sodium bisulfite solution with a final concentration of 20 mg / mL.

[0019] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the centrifugal separation in step 2) is performed at a speed of 10,000 g for 20-40 minutes.

[0020] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the filtration in step 2) is performed using a 0.22 μm filter.

[0021] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the freeze-drying in step 2) is performed at -50°C for 48 hours.

[0022] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the buffer described in step 4) is 25mM Tris-HCl buffer, pH 7.4.

[0023] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the flow rate of collecting the eluate in step 4) is 15 mL / h.

[0024] Furthermore, in the above-mentioned method for extracting and purifying potato protein with whitening and anti-aging effects, the filler of the gel chromatography column is dextran gel or agarose gel.

[0025] The last technical solution provided by the present invention is the application of the above-mentioned potato protein with whitening and anti-aging effects as an active additive in cosmetics.

[0026] Specifically, the potato protein with whitening and anti-aging effects mentioned above is added to skin care products and sunscreen products, which has a positive effect on improving skin color, preventing spots and resisting skin free radical oxidative aging.

[0027] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0028] 1. The two potato proteins (39kD and 17kD) separated and purified by the present invention are cytotoxic to cultured mouse skin melanoma cells, but non-toxic to normal human immortalized epidermal cells; they can effectively inhibit the production of melanin in skin cells, show good cell tyrosinase inhibition ability, and have a good whitening effect;

[0029] 2. The two potato proteins (39kD and 17kD) separated and purified by the present invention have strong antioxidant capacity, can remove various forms of free radicals in cells, reduce oxidative stress damage to skin cells, and delay skin aging;

[0030] 3. The technical solution provided by the present invention extracts total potato protein through the steps of precipitation, centrifugation, filtration, dialysis and freeze drying, and then separates the purified protein components through a gel chromatography column. The prepared protein has high purity and the extraction and purification method is simple;

[0031] 4. The potato protein with whitening and anti-aging effects provided by the present invention is added to skin care products and sunscreen products, which has a positive effect on improving skin color, preventing spots and resisting skin free radical oxidative aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart of the extraction and purification of potato protein components in Example 1.

[0033] Figure 2 The graph shows the potato protein concentration of the eluate collected after purification by Sephadex G-75 column chromatography.

[0034] Figure 3 The figure is the SDS-PAGE electrophoresis analysis diagram of the potato protein separated and purified by the present invention.

[0035] Wherein: M: MW marker; Lane 1: 17 kD purified protein fraction; Lane 2: 39 kD purified protein fraction; Lane 3: total protein.

[0036] Figure 4 The figure is a standard curve diagram for determining the relative molecular weights of protein components and the relative molecular weights of two protein components extracted and purified by the present invention.

[0037] Figure 5 Statistical results of in vitro cytotoxicity experiments on human immortalized epidermal cells (HaCaT) treated with different concentrations of 39kD and 17kD protein components.

[0038] Figure 6 The statistical results of in vitro cytotoxicity experiments on the melanoma cell line B16F0 after being treated with different concentrations of 39kD and 17kD protein components.

[0039] Figure 7 These are the statistical results of the experiment on the melanin content in the melanoma cell line B16F0 after being treated with different concentrations of 39kD and 17kD protein components.

[0040] Figure 8 These are the statistical results of the tyrosinase activity experiment after the melanoma cell line B16F0 was treated with different concentrations of 39kD and 17kD protein components.

[0041] Fig. 9 This is a statistical graph of the in vitro reducing power test results of the two protein components.

[0042] Fig.10 Statistical graph of the in vitro hydroxyl radical scavenging activity experimental results of the two protein components.

[0043] Fig.11 Statistical graph of the in vitro DPPH free radical scavenging activity experimental results of the two protein components. DETAILED DESCRIPTION

[0044] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0045] Example 1

[0046] This embodiment provides a method for separating and purifying potato protein components. The extraction and purification flow chart is as follows: Figure 1 As shown; specifically including the following steps:

[0047] 1) 400 g of fresh potatoes were washed, peeled and cut into pieces, 400 mL of water was added according to a material-water ratio of 1:1, and the mixture was ground using a blender to obtain 600 mL of potato juice, to which 12 g of sodium bisulfite was added to a final concentration of 20 mg / mL to prevent polyphenol oxidation;

[0048] 2) Allow the mixture to stand for 30 minutes at 4°C to allow starch to precipitate, take the supernatant, centrifuge the supernatant at 10,000 g for 30 minutes at 4°C, take the supernatant again, and filter the supernatant with a 0.22 μm filter to obtain transparent potato juice;

[0049] 3) dialyzing the potato juice prepared in step 2) at 4° C. for 3 days, freeze-drying to obtain potato total protein, and storing the extracted potato total protein in a refrigerator at -20° C. for subsequent use;

[0050] Total protein extraction rate = total protein mass of potatoes / total mass of potatoes × 100%

[0051] 4) Load potato total protein onto a gel chromatography column, pre-equilibrate with 25 mM Tris-HCl buffer (pH 7.4), elute at a flow rate of 15 mL / h, collect 100 tubes of eluate in a test tube, and use a BCA kit to determine the protein concentration in each test tube. Draw a graph of the protein concentration of the eluate in different tubes ( Figure 2 ), collect two peak components, and perform SDS-PAGE analysis on the two separated and purified components. Figure 3 ; According to the relative mobility and molecular weight logarithm of the standard protein in Table 2, a standard curve can be obtained ( Figure 4 ), according to the standard curve, the relative mobility R of the unknown protein can be used f To calculate the molecular weight M of the unknown protein. The key steps are as follows:

[0052] ① According to the instructions, the protein content of the two purified protein components was determined using a BCA protein concentration detection kit and the extraction rate of each protein component was calculated. The protein content (g) and the extraction rate obtained in each step of this embodiment are shown in Table 1.

[0053] The extraction rate of each purified protein component was calculated according to the following formula: Extraction rate of purified protein component = (mass of purified protein component / total protein mass) × 100%.

[0054] Table 1

[0055] Test product Total protein (g) Extraction rate (%) Step 3) Prepared total protein 12.80 3.90 39kD protein 0.22 1.72 17kD protein 0.20 1.56

[0056] ② The homogeneity of the purified protein components was determined by SDS-PAGE electrophoresis analysis. The purified protein components were shown on the gel ( Figure 3 ), and the single band of the purified protein fraction demonstrated its homogeneity.

[0057] ③ Calculation of molecular weight of purified protein components:

[0058] The relative mobility of proteins R f = Distance moved by protein sample (cm) / Distance moved by indicator dye (cm) The distance moved by protein sample is the distance from the top of SDS-PAGE gel to the center of protein sample band;

[0059] The distance the indicator dye migrates is the distance from the top of the SDS-PAGE gel to the dye front;

[0060] The standard curve obtained according to Table 2 is as follows Figure 4 The calculated molecular weights of the purified protein fractions are also indicated in Figure 4 In the study, they were 39kD and 17kD, respectively.

[0061] Table 2

[0062]

[0063]

[0064] In summary, SDS-PAGE showed two single bands with molecular weights of 39 kD and 17 kD, respectively; this proves that this embodiment prepared two potato protein components with molecular weights of 39 kD and 17 kD, respectively, with purities of 70% and 73%, respectively, and extraction rates of 1.72% and 1.56%, respectively.

[0065] Example 2

[0066] Study on the toxicity of 39kD and 17kD potato proteins to human immortalized epidermal (HaCaT) cells and melanoma (B16F0) cells

[0067] The cultured HaCaT cells or B16F0 cells were inoculated into 96-well plates (1×10 4Cells were cultured in 5% CO2 at 37°C for 24 hours to promote cell adhesion. Fresh culture medium containing different concentrations of potato protein (0.25, 0.5, 1, 2 or 4 mg / mL; 0 mg / mL as control) was then used for 24 hours at 37°C. 10% CCK-8 (10% CCK-8 as blank) was added and incubated for 1.5 hours under light-proof conditions, and the absorbance at 450nm was measured with an enzyme reader. Three parallel experiments were set for each concentration. Cell viability was calculated according to the following formula:

[0068]

[0069] Where A is the absorbance of the test group, A0 is the absorbance of the blank group, and A c is the absorbance of the control group.

[0070] Experimental results:

[0071] The CCK-8 method was used to detect the inhibitory effects of the two purified potato proteins on the proliferation of HaCaT cells and B16F0 cells in vitro. Figure 5-6 .pass Figure 5 The experimental results show that potato protein has no obvious toxic side effects on normal HaCaT skin cells within the test range (0.25-4mg / mL). The cell survival rate of 39kD protein at a concentration of 4mg / mL is 91.3%, and the cell survival rate of 17kD protein at a concentration of 4mg / mL is 88.5%, which can be considered "non-toxic". In contrast, the inhibitory effect on B16F0 cells was significant (p < 0.005) ( Figure 6 ), and showed concentration dependence. At the same concentration, the inhibitory effect of the 17kD protein component on B16F0 cell viability was significantly higher than that of the 39kD protein component. These results show that the 17kD protein and the 39kD protein have significant cytotoxicity to melanoma B16F0 cells, but have no irritation to skin cells HaCaT.

[0072] Example 3

[0073] Effects of two isolated and purified potato proteins on melanin content in B16F0 cells

[0074] The cultured B16F0 cells were cultured at 1×10 5The cells were inoculated into 6-well plates at a density of 10 cells / well and incubated at 37°C, 5% CO2 with or without potato protein components (0, 0.25, 0.5, 1, 2 or 4 mg / mL; 0 mg / mL as control) for 48 hours. After incubation, the cells were washed twice with PBS and harvested by centrifugation at 1200 rpm for 3 minutes at room temperature. The cells were lysed in 1M NaOH [10% (v / v) DMSO] {1M NaOH [10% (v / v) DMSO] as blank} at 80°C for 1 hour. Immediately after lysis, the absorbance of the sample at 405 nm was determined by spectrophotometry, and three parallel experiments were set for each concentration. The relative melanin content in B16F0 cells was calculated according to the following formula and expressed as the relative melanin content of the cells:

[0075]

[0076] Where A t is the absorbance of the test group, A0 is the absorbance of the blank group, A c is the absorbance of the control group.

[0077] Experimental results:

[0078] In order to further evaluate whether potato protein can reduce the content of melanin in cells and verify its whitening effect, the melanin content of B16F0 cells under the action of different concentrations of potato protein was measured. Figure 7 It can be seen that within the concentration range of 0.25-4mg / mL, both purified potato proteins can reduce the production of melanin in cells. And the melanin content in B16F0 cells treated with 17kD protein is significantly lower than that in B16F0 cells treated with 39kD protein at the same concentration. When the treatment concentration of 39kD protein and 17kD protein is 4mg / mL, the relative melanin content in B16F0 cells is 34.6% and 31.2%, respectively, both of which are reduced to less than 50% of the control group, showing a significant inhibitory effect on melanin production.

[0079] Example 4

[0080] Effects of two isolated and purified potato proteins on tyrosinase activity in B16F0 cells

[0081] Tyrosinase activity was evaluated by measuring the oxidation of levodopa (L-DOPA). B16F0 cells were cultured in the same manner as above, with or without protein components (0, 0.25, 0.5, 1, 2 or 4 mg / mL; 0 mg / mL as control) at 37°C and 5% CO2 for 48 h, and the cells were washed twice with cold PBS, followed by addition of PBS (50 μL) containing 0.1% Triton X-100 and lysis at -80°C for 30 min. Then 150 μL of the reaction mixture containing 0.1% L-DOPA was added to the samples in the 96-well plate, incubated at 37°C for 1 h, and the sample absorbance was measured at 475 nm. Three parallel experiments were set up for each concentration. The tyrosinase activity of B16F0 cells was calculated according to the following formula and expressed as relative cellular tyrosinase activity:

[0082]

[0083] Where A t A0 is the absorbance of the experimental group, and A1 is the absorbance of the control group.

[0084] Experimental results:

[0085] Tyrosinase is a key enzyme that regulates melanin production in cells. Figure 8 It can be seen that with the increase of potato protein concentration, both potato proteins can significantly inhibit the activity of tyrosinase in B16F0 cells. The relative tyrosinase activity of 39kD protein at 2mg / mL and 4mg / mL concentrations was 73.7% and 68.2%, respectively. In comparison, 17kD protein showed better tyrosinase inhibition at 2mg / mL and 4mg / mL concentrations, with relative tyrosinase activity of 68.5% and 53%, respectively. The results show that both potato proteins reduce the content of cellular melanin through the tyrosinase inhibition mechanism.

[0086] Example 5

[0087] Three in vitro antioxidant activity tests (reducing power, hydroxyl radical and DPPH radical scavenging ability) of two isolated and purified potato proteins.

[0088] 1) Reduction power

[0089] 1 mL of potato protein (0.25, 0.5, 1, 2 or 4 mg / mL), PBS (pH 7.4) and 1% (m / m) potassium ferricyanide solution were taken in a 10 mL centrifuge tube. After 20 minutes of constant temperature water bath at 50°C, 1 mL of 10% (m / m) trichloroacetic acid solution was added to the centrifuge tube. After oscillation and mixing, centrifuge at 4000 rpm for 10 minutes. Then 1 mL of the supernatant after centrifugation was mixed with 1 mL of distilled water and placed in a new centrifuge tube. 1 mL of 0.1% (m / m) FeCl3 solution was added, oscillation and mixing were performed. After standing for 10 minutes, the absorbance A was measured at 700 nm. s PBS was used as the blank group to replace the two potato proteins and the absorbance was A b Three parallel experiments were performed for each concentration. The reducing power was calculated according to the following formula:

[0090] Reducing power (%) = (A s -A b ) / A b ×100%

[0091] Experimental results:

[0092] The results of the in vitro reducing power of two potato proteins refer to Fig. 9 In the range of mass concentration of 0.25-4 mg / mL, the 39kD protein and the 17kD protein had a significant effect on Fe 3+ It showed good reducing power, and its reducing power increased with the increase of concentration. At 4 mg / mL, the reducing power of 39kD protein was 38.6%, while that of 17kD protein was 56.5%, and that of OVA was 42.9%. Within the concentration range tested, although the reducing power of the two potato proteins was weaker than that of vitamin C (Vc), they both showed good reducing power, among which the performance of 17kD protein was significantly better than that of OVA, while that of 39kD protein was slightly weaker than that of OVA.

[0093] 2) Hydroxyl free radical scavenging rate

[0094] 1 mL of potato protein of different concentrations (0.25, 0.5, 1, 2 or 4 mg / mL), FeSO4 solution (9 mM), ethanol-salicylic acid solution (9 mM) and H2O2 (8.8 mM) were added to a 10 mL centrifuge tube, mixed evenly, and placed in a 37°C water bath for 20 min. After centrifugation at 4000 rpm for 10 min, the absorbance A was measured at 510 nm. x Distilled water was used to replace the sample and H2O2 solution was used as blank and control group, and the absorbance was measured as A x0 and A0. Three parallel experiments were set for each concentration. The hydroxyl radical scavenging rate was calculated according to the following formula:

[0095] Hydroxyl radical scavenging rate (%) = (A0 + A x0 -A x ) / A0×100%

[0096] Experimental results:

[0097] The scavenging ability of two potato proteins on hydroxyl free radicals Fig.10 In the range of mass concentration of 0.25-4mg / mL, the two potato proteins showed a certain ability to scavenge hydroxyl radicals, and their scavenging abilities increased slowly with the increase of protein concentration. At 4mg / mL, the hydroxyl radical scavenging rate of 39kD protein was 23.5%, the hydroxyl radical scavenging rate of 17kD protein was 27.6%, and the hydroxyl radical scavenging rate of control OVA protein was 26.9%. Fig.10 It can be seen that within the tested concentration range, the free radical scavenging ability of 17kD protein was always higher than that of OVA protein and lower than that of Vc, while the free radical scavenging ability of 39kD protein was slightly lower than that of OVA protein.

[0098] 3) DPPH free radical scavenging rate

[0099] Take 2mL of potato protein solution with different concentrations (0.25, 0.5, 1, 2 or 4mg / mL) and 2mL of 0.02g / L DPPH ethanol solution, add them into a 10mL centrifuge tube in sequence, mix well and quickly place at 25℃ in the dark for 30min. Measure the absorbance A at 517nm x , anhydrous ethanol was used to replace the sample and DPPH solution as blank and control group, and the absorbance was measured as A x0 and A0. Three parallel experiments were set for each concentration. The DPPH free radical scavenging rate was calculated according to the following formula:

[0100] DPPH free radical scavenging rate (%) = (A0 + A x0 -A x ) / A0×100%

[0101] Experimental results:

[0102] The results of the DPPH free radical scavenging ability of two potato proteins are shown in Fig.11, within the mass concentration range of 0.25-4mg / mL, its DPPH free radical scavenging ability increased with the increase of mass concentration. At 4mg / mL, the DPPH free radical scavenging rate of 39kD protein was 65.0%, the DPPH free radical scavenging rate of 17kD protein was 85.0%, and the DPPH free radical scavenging rate of control OVA protein was 80.7%. Within the concentration range tested, the DPPH free radical scavenging ability of 17kD protein was always higher than OVA protein and lower than Vc, and was only slightly lower than Vc at high concentrations; while the free radical scavenging ability of 39kD protein was slightly lower than that of OVA protein.

[0103] Table 3 Half inhibition concentration (IC) of two potato protein components, OVA protein and Vc in different oxidation systems 50 value)

[0104]

[0105] Note 1: Vc has strong antioxidant capacity, --- indicates a negative value;

[0106] Note 2: The values ​​in the table are the average values ​​of three measurements, and ± is the standard deviation.

[0107] Experimental results:

[0108] By comparing the half inhibition concentration (IC) of two potato proteins in different antioxidant evaluation systems 50 The values ​​(see Table 3) show that the two potato proteins showed the strongest scavenging ability against DPPH free radicals (39kD protein IC 50 The value is 1.97±0.30mg / mL, 17kD protein IC 50 The value was 0.64±0.04mg / mL), and it showed good reducing power for trivalent iron ions (39kD protein IC 50 The value was 12.63±2.62mg / mL, and the IC value of 17kD protein was 50 The value is 3.13±0.28mg / mL), and the scavenging ability of hydroxyl free radicals is slightly weak (39kD protein IC 50 The value is 209.50±5.90mg / mL, 17kD protein IC 50 value was 104.43±2.67mg / mL); at the same time, within the test range, the reducing power, hydroxyl radical and DPPH radical scavenging abilities of the 17kD protein were significantly stronger than those of the 39kD protein. Although lower than Vc, its reducing power and DPPH radical scavenging abilities were significantly stronger than those of the OVA protein control group.

[0109] In summary, the present invention provides potato protein with whitening and anti-aging effects and its extraction and purification method. The two purified potato proteins show good cell tyrosinase activity inhibition ability, can significantly reduce the melanin content in cells, and have a good whitening effect. At the same time, the two potato proteins also show good antioxidant activity, can effectively remove free radicals, thereby protecting cells from oxidative stress, and have skin antioxidant and anti-aging effects. At the same time, the present invention also discloses an extraction and purification method for purified potato protein, firstly extracting total protein through steps such as precipitation, centrifugation, filtration, dialysis and freeze drying, and then separating through a gel chromatography column to obtain two purified protein components. The present invention is the first to propose a potato protein component with whitening and anti-aging effects and its extraction and purification method, and to study and compare the antioxidant stress ability of potato proteins of different molecular weights, as well as their effects on melanoma cell proliferation ability, melanin production and tyrosinase activity, which provides new ideas and new directions for the development of cosmetic active ingredients with whitening and anti-aging effects.

[0110] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Potato protein with whitening and anti-aging effects, characterized in that: The molecular weight of the potato protein is 39kD and 17kD.

2. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The steps are as follows: 1) washing, peeling and cutting fresh potatoes, grinding them with water, and then adding polyphenol antioxidants to obtain a mixture; 2) at 3-5° C., the mixture prepared in step 1) is allowed to stand to allow starch to precipitate, the supernatant is collected, centrifuged, the supernatant is collected again, and filtered to obtain transparent potato juice; 3) dialyzing the potato juice prepared in step 2) at 3-5° C. for 2-4 days, freeze-drying to obtain total potato protein, and storing it in a refrigerator at -20° C. for later use; 4) The potato total protein is separated and purified by gel chromatography column, pre-balanced with buffer, the eluate is collected, and the eluate in each tube is freeze-dried to obtain a purified protein component.

3. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The mass ratio of potatoes to water in step 1) is 1:

1.

4. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The polyphenol antioxidant described in step 1) is a sodium bisulfite solution with a final concentration of 20 mg / mL.

5. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The centrifugal separation in step 2) is performed at a speed of 10,000 g for 20-40 minutes; the filtration in step 2) is performed using a 0.22 μm filter.

6. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The freeze drying in step 2) is performed at -50°C for 48 hours.

7. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The buffer described in step 4) is 25 mM Tris-HCl buffer, pH 7.

4.

8. The method for extracting and purifying potato protein with whitening and anti-aging effects according to claim 1, characterized in that: Step 4) The flow rate of the eluent collection was 15 mL / h.

9. The potato protein with whitening and anti-aging effects according to claim 1, characterized in that: The filler of the gel chromatography column is dextran gel or agarose gel.

10. Use of the potato protein with whitening and anti-aging effects as claimed in claim 1 as an active additive in cosmetics.

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