Polypeptide and application thereof in preparation of whitening product

Abstract

The invention provides a polypeptide and application thereof in preparation of a whitening product. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1, and the polypeptide can effectively inhibit the activity of tyrosinase and is suitable for preparing a tyrosinase inhibitor; generation of melanin can be remarkably inhibited, and the method is suitable for preparing melanin inhibitors; moreover, the antioxidant activity is excellent, and the antioxidant is suitable for preparing antioxidant products. Therefore, the polypeptide disclosed by the invention is very suitable for preparing whitening products.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and more specifically, relates to a polypeptide and its application in the preparation of whitening products. Background Art

[0002] With the increase of ultraviolet radiation and work pressure, people are more likely to have problems such as dull skin and increased spots. In order to improve skin color, the commonly used methods are chemical whitening, physical whitening, natural whitening, etc. Chemical whitening is the use of cosmetics containing chemical whitening ingredients such as arbutin to inhibit the production of melanin. Physical whitening is the use of photorejuvenation, laser whitening and other means to remove melanin from the surface of the skin, but both methods are irritating and should not be used for a long time. Natural whitening is the use of cosmetics containing natural whitening ingredients such as pearl powder, but the whitening effect of natural whitening ingredients is relatively weak and takes a long time to take effect.

[0003] Peptides have received considerable attention in the development of whitening products due to their high safety and rapid effectiveness, but the existing peptide resources that can be used for whitening are still relatively scarce. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, enrich the resource library of polypeptides with whitening effects, and provide more raw material options for whitening products.

[0005] The first object of the present invention is to provide a polypeptide.

[0006] The second object of the present invention is to provide the use of the above polypeptide in the preparation of whitening products.

[0007] The third object of the present invention is to provide the use of the above polypeptide in the preparation of tyrosinase inhibitors.

[0008] The fourth object of the present invention is to provide the use of the above polypeptide in the preparation of a melanin inhibitor.

[0009] The fifth object of the present invention is to provide the use of the above polypeptide in the preparation of antioxidant products.

[0010] The sixth object of the present invention is to provide polypeptide-related biological materials.

[0011] The seventh objective of the present invention is to provide the use of the above-mentioned related biomaterials in the preparation of whitening products.

[0012] The eighth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of tyrosinase inhibitors.

[0013] The ninth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of melanin inhibitors.

[0014] The tenth objective of the present invention is to provide the use of the above-mentioned related biological materials in the preparation of antioxidant products.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1. SEQ ID NO: 1: IVHRKCF.

[0017] The functions of the above polypeptides are as follows: (1) they can effectively inhibit the activity of tyrosinase and are suitable for preparing tyrosinase inhibitors; (2) they can significantly inhibit the production of melanin and are suitable for preparing melanin inhibitors; (3) they have excellent antioxidant activity and are suitable for preparing antioxidant products.

[0018] In summary, the above polypeptides are also suitable for preparing whitening products. Therefore, the use of the above polypeptides in preparing whitening products, tyrosinase inhibitors, melanin inhibitors, and antioxidant products should be within the protection scope of the present invention.

[0019] Based on this, the present invention also provides related biological materials of the above-mentioned polypeptide and their use in the preparation of whitening products, tyrosinase inhibitors, melanin inhibitors, and antioxidant products. The related biological materials are nucleic acid molecules that can express the polypeptide, or recombinant DNA, expression cassettes, transposons, vectors or host cells containing the nucleic acid molecules.

[0020] In addition, the present invention also provides a product comprising the above polypeptide and / or its related biological materials.

[0021] Preferably, the relevant biological material is a nucleic acid molecule capable of expressing the polypeptide, or a recombinant DNA, an expression cassette, a transposon, a vector or a host cell containing the nucleic acid molecule.

[0022] Preferably, the product is food, cosmetic or medicine.

[0023] More preferably, it also contains excipients acceptable to food, cosmetics or medicines.

[0024] Further preferably, the food is nutritious and healthy food.

[0025] The present invention has the following beneficial effects:

[0026] The polypeptide of the present invention can not only effectively inhibit the activity of tyrosinase, but also be suitable for preparing tyrosinase inhibitors; it can also significantly inhibit the production of melanin, and is suitable for preparing melanin inhibitors; and it has excellent antioxidant activity, and is suitable for preparing antioxidant products. Therefore, the polypeptide of the present invention is very suitable for preparing whitening products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The purity of the peptide was identified.

[0028] Figure 2 The mass spectrometry identification results of the peptides.

[0029] Figure 3 The toxicity test results of the peptides.

[0030] Figure 4 This is a Lineweaver-Burk diagram.

[0031] Figure 5 This is the corresponding relationship between the slope of the straight line and the peptide concentration.

[0032] Figure 6 This is a graph showing the results of measuring tyrosinase activity after the action of polypeptide.

[0033] Figure 7 This is a graph showing the results of the determination of melanin content after the action of polypeptides.

[0034] Figure 8 This is a graph showing the results of measuring SOD activity after the action of polypeptide.

[0035] Fig. 9 This is a graph showing the results of measuring the activity of GSH-PX after the action of polypeptide.

[0036] Fig.10 This is a graph showing the results of determining the relative activity of ROS after the action of polypeptides. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] All experiments in the embodiments of the present invention were set up in three groups of parallel experiments, and the final data were expressed as mean ± standard deviation. The statistical graphs were completed under Origin 2019 software, and the experimental results were analyzed by one-way analysis of variance on the means of each group using IBM SPSS Statistics 26 software. P < 0.05 indicates that the difference is statistically significant.

[0040] Example 1 Synthesis and identification of polypeptides

[0041] Shanghai Aminlian Biotechnology Co., Ltd. was commissioned to assist in the synthesis of the polypeptide (the amino acid sequence is shown in SEQ ID NO: 1: IVHRKCF), and the purity and mass spectrometry of the polypeptide were performed.

[0042] 1. Purity Identification

[0043] The purity of the peptide was identified by high performance liquid chromatography.

[0044] Among them, the identification conditions of high performance liquid chromatography are:

[0045] Chromatographic column: Inertsil ODS-SP (4.6×250 mm);

[0046] Mobile phase A: water (containing 0.1% (v / v) trifluoroacetic acid);

[0047] Mobile phase B: acetonitrile (containing 0.1% (v / v) trifluoroacetic acid);

[0048] Elution gradient: 0→25 min, 99% (v / v)→50% (v / v) A;

[0049] Flow rate: 1 mL / min;

[0050] Detection wavelength: 220nm;

[0051] Sample volume: 30 μL.

[0052] The results of purity identification are as follows Figure 1 As shown, it can be seen that the main peak in the polypeptide of this example accounts for the majority, with only very few impurity peaks appearing, and the purity is as high as 96.86%.

[0053] 2. Mass spectrometry identification

[0054] ESI-MS mass spectrometry was used to identify the molecular weight of the peptide.

[0055] Among them, the identification conditions of ESI-MS mass spectrum are:

[0056] Mobile phase A: water (containing 0.1% (v / v) trifluoroacetic acid);

[0057] Mobile phase B: acetonitrile (containing 0.1% (v / v) trifluoroacetic acid);

[0058] Flow rate: 2mL / min;

[0059] Running time: 4 minutes;

[0060] Operation mode: positive ion mode;

[0061] Scanning range: 0~2000Da.

[0062] The results of mass spectrometry identification are as follows Figure 2 As shown, it can be seen that the molecular ion peak m / z of the polypeptide of this example is 903.26, with one charge, which is consistent with its theoretical molecular weight value, indicating that the polypeptide synthesized in this example is the polypeptide with the amino acid sequence shown in SEQ ID NO: 1.

[0063] Example 2 The polypeptide is non-toxic to cells

[0064] 1. Solution preparation

[0065] Sample solution: In a clean bench, the polypeptide obtained in Example 1 was dissolved in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin dual antibody to a final concentration of 100, 200, and 400 μM, respectively, and then sterilized with a 0.22 μm syringe filter.

[0066] Kojic acid solution: In a clean bench, dissolve kojic acid in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody to a final concentration of 300 μM, and then sterilize with a 0.22 μm syringe filter.

[0067] 2. Experimental Grouping and Treatment

[0068] Dosing sample hole (A 样 ): Melanoma cells (B16F10) in the logarithmic growth phase were inoculated in a 96-well plate at a density of 3000 cells / well, and adaptively cultured in a 37°C incubator for 24 hours, then the supernatant was discarded, 100 μL of sample solution / kojic acid solution were added, and cultured in a 37°C incubator for 24 hours, then the supernatant was discarded, 110 μL of 10% (v / v) CCK-8 solution was added under light-proof conditions, and the plates were incubated in a 37°C incubator for 30 minutes, and the absorbance of each well at 450 nm was measured.

[0069] Blank well (A 空 ): Add 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody to a 96-well plate, culture in an incubator at 37°C for 24 hours, then discard the supernatant, add 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody, culture in an incubator at 37°C for 24 hours, then discard the supernatant, add 110 μL of 10% (v / v) CCK-8 solution under light-proof conditions, incubate in an incubator at 37°C for 30 minutes, and measure the absorbance of each well at 450 nm.

[0070] Control wells (A 0): Melanoma cells (B16F10) in the logarithmic growth phase were inoculated in a 96-well plate at a density of 3000 cells / well, and adaptively cultured in a 37°C incubator for 24 hours, then the supernatant was discarded, 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody was added, and cultured in a 37°C incubator for 24 hours, then the supernatant was discarded, 110 μL of 10% (v / v) CCK-8 solution was added under light-proof conditions, and the plates were incubated in a 37°C incubator for 30 minutes, and the absorbance of each well at 450 nm was measured.

[0071] According to the formula "Cell viability (%) = [(A 样 -A 空 ) / (A 0 -A 空 )]×100%” to calculate the cell viability after the action of sample solution / kojic acid solution to evaluate the toxicity of the peptide.

[0072] 3. Experimental Results

[0073] The results are as follows Figure 3 As shown, compared with the blank wells, there was no significant difference in the activity of B16F10 cells after the action of 100-400 μM polypeptides, indicating that the polypeptide of the present invention is non-toxic to cells and has high safety.

[0074] Example 3: Peptides can inhibit tyrosinase activity in vitro

[0075] 1. Solution preparation

[0076] Sample solution: The polypeptide obtained in Example 1 was dissolved in a phosphate buffer at pH = 6.8 to a final concentration of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively.

[0077] L-Tyrosine solution: L-Tyrosine was dissolved in phosphate buffer at pH 6.8 to a final concentration of 0.5 mg / mL.

[0078] Tyrosinase solution: Dissolve tyrosinase in phosphate buffer at pH 6.8 to a final concentration of 500 U / mL.

[0079] 2. Experimental Grouping and Treatment

[0080] Solvent blank well (A1): Add 40 μL of PBS solvent into the 96-well plate.

[0081] Solvent background well (A2): Add 40 μL of L-tyrosine solution to the 96-well plate.

[0082] Sample background well (A3): Add 40 μL of sample solution into the 96-well plate.

[0083] Sample reaction well (A4): Add 40 μL of L-tyrosine solution and 40 μL of sample solution to the 96-well plate.

[0084] The total solution system was 110 μL (complemented with PBS solvent). After incubation at 37°C for 15 min, 20 μL of tyrosinase solution was added to each well, and then incubated at 37°C for 10 min. The absorbance value was measured at 475 nm, and the tyrosinase inhibition rate was calculated according to the formula "Tyrosinase inhibition rate (%) = [1-(A4-A3) / (A2-A1)] × 100%".

[0085] 3. Experimental Results

[0086] The results showed that the IC 50 The value is 0.80±0.03 mM, indicating that the polypeptide of the present invention can significantly inhibit the activity of tyrosinase and is suitable for preparing whitening products.

[0087] Example 4 The inhibitory type of the polypeptide on tyrosinase is mixed inhibition

[0088] 1. Solution preparation

[0089] Sample solution: The polypeptide obtained in Example 1 was dissolved in a phosphate buffer at pH 6.8 to a final concentration of 0, 0.10, 0.15, and 0.20 mM, respectively.

[0090] L-tyrosine solution: L-tyrosine was dissolved in phosphate buffer at pH 6.8 to a final concentration of 0.75, 1.00, 1.25, 2.00, and 3.00 mM, respectively.

[0091] Tyrosinase solution: Dissolve tyrosinase in phosphate buffer at pH 6.8 to a final concentration of 500 U / mL.

[0092] 2. Experimental Grouping and Treatment

[0093] In a 96-well plate, 40 μL of L-tyrosine solution, 40 μL of sample solution and 30 μL of phosphate buffer (pH = 6.8) were added, and incubated at 37°C for 10 min. Then, 20 μL of tyrosinase solution was added, and the plate was incubated at 37°C for another 10 min. The absorbance at 475 nm was measured at 0 and 10 min of the incubation. According to the formula "reaction rate (%) = (A 10 -A 0 ) / 10]×100%”(A 10 is the absorbance value at 10 min, A 0The reaction rate between the peptide and tyrosinase was calculated using the absorbance value at 0 min. A Lineweaver-Burk plot was made based on the calculation results. Figure 4 As shown, and according to Figure 4 The corresponding relationship between the slope of each straight line and the peptide concentration is shown in Figure 5 .

[0094] 3. Experimental Results

[0095] Depend on Figure 4 It can be seen that the maximum reaction rate (Vmax, the reciprocal of the intercept on the y-axis) and the Michaelis constant (Km, the reciprocal of the intercept on the x-axis) in the coordinate graph change with the change of the polypeptide concentration, and the four straight lines all intersect in the second quadrant of the coordinate axis, indicating that the inhibition of the polypeptide on tyrosinase is a mixed result of competition and non-competitiveness, that is, the inhibition type of the polypeptide of the present invention on tyrosinase is mixed inhibition.

[0096] Depend on Figure 5 It can be seen that the slope of each straight line in the Lineweaver-Burk plot is linearly related to the polypeptide concentration, indicating that the polypeptide of the present invention can bind to a single site on tyrosinase.

[0097] Example 5 Polypeptide can inhibit tyrosinase activity at the cellular level

[0098] 1. Solution preparation

[0099] Sample solution: The polypeptide obtained in Example 1 was dissolved in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin dual antibody to a final concentration of 100 and 200 μM, respectively.

[0100] Kojic acid solution: dissolve kojic acid in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody to a final concentration of 300 μM.

[0101] 2. Experimental Grouping and Treatment

[0102] Sample measurement hole (A 测 ): Melanoma cells (B16F10) in the logarithmic growth phase were cultured at 5×10 5 The cells were seeded at a density of 10 cells / well in a 6-well plate. After adaptive culture in a 37°C incubator for 24 hours, the supernatant was discarded and washed twice with PBS solution (pH = 7.4). 2 mL of sample solution / kojic acid solution were added respectively, and the cells were cultured in a 37°C incubator for 24 hours. The supernatant was discarded, washed twice with PBS solution (pH = 7.4) to remove non-adherent cells, and 400 μL of lysis buffer (PMSF: RIPA volume ratio is 1:100) was added respectively. The cells were lysed at 4°C for 30 minutes and the supernatant was collected by centrifugation.

[0103] Blank well (A 空 ): The same sample measurement wells were used, except that 2 mL of sample solution was replaced with an equal volume of DMEM culture medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody.

[0104] After the protein concentration of the supernatant was determined by BCA kit and normalized, 50 μL of protein solution was taken and mixed with 50 μL of PBS and 50 μL of L-DOPA (10 mM). After reacting at 37°C for 30 min, the absorbance was measured at 475 nm. According to the formula "Tyrosinase activity (%) = A 测 / A 空 × 100%” to calculate the tyrosinase activity after the sample solution / kojic acid solution was used.

[0105] 3. Experimental Results

[0106] The results are as follows Figure 6 As shown, compared with the blank wells, the tyrosinase activity of B16F10 cells was significantly reduced after the action of 100-200 μM polypeptide, and was even lower than the tyrosinase activity after the action of kojic acid, indicating that the polypeptide of the present invention can significantly inhibit the activity of tyrosinase and is suitable for preparing whitening products.

[0107] Example 6: Peptide can inhibit the production of melanin

[0108] 1. Solution preparation

[0109] Sample solution: In a clean bench, the polypeptide obtained in Example 1 was dissolved in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin dual antibody to a final concentration of 100 and 200 μM, respectively, and then sterilized with a 0.22 μm syringe filter.

[0110] Kojic acid solution: In a clean bench, dissolve kojic acid in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody to a final concentration of 300 μM, and then sterilize with a 0.22 μm syringe filter.

[0111] 2. Experimental Grouping and Treatment

[0112] Sample measurement hole (A 测 ): Melanoma cells (B16F10) in the logarithmic growth phase were cultured at 5×10 5The cells were inoculated at a density of 10 cells / well in a 6-well plate, and after adaptive culture in an incubator at 37°C for 24 hours, the supernatant was discarded and washed twice with PBS solution (pH=7.4), 2 mL of sample solution / kojic acid solution were added respectively, and after culture in an incubator at 37°C for 24 hours, the supernatant was discarded, and the cells were washed twice with PBS solution (pH=7.4) to remove non-adherent cells, and then the cells were digested with trypsin, the digestion products were collected and centrifuged at 1500 r / min for 10 minutes, 1 mL of NaOH solution (1 mol / L) containing 10% (v / v) DMSO was added to the precipitate obtained by centrifugation, and after extraction in a water bath at 80°C for 1 hour, the absorbance value of each well at 450 nm was measured.

[0113] Blank well (A 空 ): The same sample measurement wells were used, except that 2 mL of sample solution was replaced with an equal volume of DMEM culture medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody.

[0114] According to the formula "Melanin content (%) = A 测 / A 空 × 100%” to calculate the melanin content after the sample solution / kojic acid solution was applied.

[0115] 3. Experimental Results

[0116] The results are as follows Figure 7 As shown, compared with the blank wells, the melanin content of B16F10 cells was significantly reduced after the action of 100-200 μM polypeptide, and was even lower than the melanin content after the action of kojic acid, indicating that the polypeptide of the present invention can significantly inhibit the formation of melanin and is suitable for preparing whitening products.

[0117] In addition, after treatment with 100 and 200 μM of the polypeptide, the melanin content of B16F10 cells was 75.34% and 61.87%, respectively, and the latter was significantly lower than the former, indicating that the inhibitory effect of the polypeptide of the present invention on melanin content was dose-dependent; Figure 6 It can be seen that the inhibitory effect of the polypeptide of the present invention on tyrosinase activity is not dose-dependent. Figure 6 and Figure 7 It can be seen that the inhibitory effect of the polypeptide of the present invention on melanin content is not simply dependent on the inhibition of tyrosinase activity.

[0118] Example 7 Peptides can effectively exert antioxidant activity

[0119] 1. Solution preparation

[0120] Sample solution: In a clean bench, the polypeptide obtained in Example 1 was dissolved in DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin dual antibody to a final concentration of 0, 100, and 200 μM, respectively, and then sterilized with a 0.22 μm syringe filter.

[0121] 2. Experimental Grouping and Treatment

[0122] (1) Determination of SOD and GSH-PX activity

[0123] Melanoma cells (B16F10) in logarithmic growth phase were cultured at 5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / well. After adaptive culture in a 37°C incubator for 24 hours, the supernatant was discarded and washed twice with PBS solution (pH = 7.4). 2 mL of sample solution was added. After culture in a 37°C incubator for 24 hours, the supernatant was discarded and washed twice with PBS solution (pH = 7.4) to remove non-adherent cells. Then 400 μL of lysis buffer (PMSF: RIPA volume ratio is 1:100) was added respectively, lysed at 4°C for 30 minutes, and the supernatant was collected by centrifugation. The activities of SOD and GSH-PX in the supernatant were determined according to the instructions of the SOD and GSH-PX kits of Nanjing Jiancheng Bioengineering Institute.

[0124] (2) ROS activity determination

[0125] Sample measurement hole (A 测 ): Melanoma cells (B16F10) in the logarithmic growth phase were seeded in a 96-well plate at a density of 3000 cells / well, and adaptively cultured in a 37°C incubator for 24 h. The supernatant was discarded and washed twice with PBS solution (pH = 7.4). 100 μL of sample solution was added and incubated at 37°C and 5% CO 2 After intervention in the incubator for 24 hours, the culture medium was discarded, and 100 μL of DCFH-DA diluted to a concentration of 10 μM with DMEM culture medium was added. After incubation in an incubator at 37°C for 30 minutes, the supernatant was discarded, and the cells were washed with PBS solution (pH = 7.4), and the absorbance value was measured with an enzyme marker (the excitation wavelength was set to 488 nm and the emission wavelength was set to 525 nm).

[0126] Blank well (A 空 ): The same sample assay wells were used, except that 100 μL of the sample solution was replaced with an equal volume of DMEM medium containing 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin double antibody.

[0127] According to "ROS relative activity (%) = A 测 / A 空 × 100%” to calculate the relative activity of ROS.

[0128] 3. Experimental Results

[0129] SOD activity results are as follows Figure 8 As shown, the GSH-PX activity results are as follows Fig. 9 The relative activity of ROS is shown in Fig.10 As shown, compared with the blank group, after the action of 100-200 μM polypeptide, the SOD and GSH-PX activities of B16F10 cells were significantly improved, and the relative activity of ROS was significantly reduced, indicating that the polypeptide of the present invention has excellent antioxidant activity and is suitable for preparing whitening products.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in the preparation of whitening products.

3. Use of the polypeptide according to claim 1 in the preparation of tyrosinase inhibitors.

4. Use of the polypeptide according to claim 1 in the preparation of a melanin inhibitor.

5. Use of the polypeptide according to claim 1 in the preparation of antioxidant products.

6. The biological material related to the polypeptide according to claim 1, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the polypeptide, or a recombinant DNA, an expression cassette, a transposon, a vector or a host cell containing the nucleic acid molecule.

7. Use of the relevant biological material described in claim 6 in the preparation of whitening products.

8. Use of the relevant biological material according to claim 6 in the preparation of tyrosinase inhibitors.

9. Use of the relevant biological material according to claim 6 in the preparation of a melanin inhibitor.

10. Use of the relevant biological material according to claim 6 in the preparation of antioxidant products.

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