A royal jelly protease hydrolysate with melanin inhibitory activity, its preparation and application

Abstract

The present invention belongs to the technical field of deep processing of agricultural products, and provides a royal jelly protease hydrolysate with melanin inhibition activity, its preparation and application. A soluble royal jelly protein solution is enzymatically hydrolyzed with pepsin to obtain an enzymatic hydrolysate; the enzymatic hydrolysate is ultrafiltered and separated to obtain a polypeptide solution with a molecular weight less than 3 KDa, namely the royal jelly protease hydrolysate. The present invention uses royal jelly as a raw material, extracts royal jelly protein, and prepares a low-molecular-weight royal jelly protease hydrolysate by using a protease enzymatic hydrolysis process and an ultrafiltration separation technology. This royal jelly protease hydrolysate has good melanin generation inhibition activity, and can reduce the generation of melanoma cells and zebrafish melanin in mice. This inhibitor is derived from the natural food royal jelly, which is green and safe, and the preparation process has controllable conditions, convenient extraction, and does not require artificial synthesis, and is expected to be applied to drugs or cosmetics with whitening effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of deep processing of agricultural products, and relates to a preparation method of royal jelly protein hydrolysate with melanin inhibitory activity. Background Art

[0002] Disclosing the information of this background art is intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Royal jelly is a jelly-like substance secreted by the hypopharyngeal glands and mandibular glands of honeybee workers. It has biological activities such as immunomodulation, antibacterial, antioxidant, and anti-aging effects, and is widely used in the fields of health products, cosmetics, etc. Royal jelly is rich in proteins, and its protein content accounts for about 9-18% of fresh royal jelly and 30%-50% of the dry matter of royal jelly. It is an important active ingredient of royal jelly and has biological activities such as antioxidant, antibacterial, anti-inflammatory, immunomodulation, wound healing, and anti-aging effects. Although royal jelly proteins have significant biological activities, they have the disadvantages of large molecular weight, being difficult to be absorbed and utilized by the human skin, and being prone to causing allergies. Therefore, increasing the absorption rate and efficacy of royal jelly proteins and reducing allergic reactions are of great significance for the development of its products. Proteolytic technology can break down proteins into small peptide segments, which have the characteristics of high safety, strong biological activity, easy absorption, and high target specificity compared with the original proteins, and can effectively enhance the biological activity and product added value of royal jelly proteins.

[0004] The hydrolysates of royal jelly proteins will obtain small peptide segments with different functions due to differences in the preparation process. For example, CN113647591 A discloses a royal jelly hydrolysate, which is a hydrolysate obtained by enzymatically hydrolyzing royal jelly with protease M-P001; the protease M-P001 is derived from Mycobacterium tuberculosis ( Mycobacterium tuberculosis ). The enzymatic hydrolysis conditions are as follows: enzymatically hydrolyze the royal jelly aqueous solution with protease M-P001, and the concentration of royal jelly in the royal jelly aqueous solution is 5-20%; add protease M-P001 at an addition amount of 1500-2500 U / g (calculated based on the royal jelly raw material), pH 6.0-8.0, and enzymatically hydrolyze at 33-36 °C for 20-25 hours. This royal jelly hydrolysate can significantly improve the clearance ability of mouse monocytes-macrophages and the activity of mouse NK cells, and has the effect of enhancing immunity activity. CN 115806588 A discloses a small peptide with the amino acid sequence IIPFIF, which is obtained by enzymatically hydrolyzing royal jelly with acid protease. This small peptide has tyrosinase inhibitory activity, and the IC of tyrosinase 50It is 6.16 mg / mL. CN 116064708 A discloses that royal jelly polypeptide and three oligopeptides, FDRIW, YPDWSW, and WHDKIF, all have the function of inhibiting oxidative stress. The royal jelly solution is reacted with acid protease at 30 - 70 °C for 3 - 8 hours to obtain an enzymatic hydrolysate; the enzymatic hydrolysate is separated and eluted through a G-15 Sephadex gel chromatography column, and a total of 5 elution peaks appear. Collect any one or more of the eluates in F1 - F5, and freeze-dry to obtain royal jelly polypeptide. Qian Jiale (Research on the Anti-Photoaging Activity of Royal Jelly and Its Enzymatic Hydrolysates [D]. Zhejiang University, 2021) dialyzed to obtain the main royal jelly protein, and used trypsin and pepsin to enzymatically hydrolyze royal jelly and the main royal jelly protein. The sample was freeze-dried to form a freeze-dried powder, and the above-mentioned royal jelly enzymatic hydrolysate was detected through a photoaging model of human fibroblasts and showed certain anti-photoaging activity. In the research of Zhu Zuoyi et al. (Zhu Zuoyi, Zhang Yu, Wang Junhong, et al. Preparation of Royal Jelly Protein Peptides and Their Hypoglycemic and Antioxidant Activities [J]. Science and Technology of Food Industry, 2020, 41(17):7), using acid protease as the enzyme preparation, with an enzyme addition amount of 6000 U / g, an enzymatic hydrolysis temperature of 43 °C, an enzymatic hydrolysis pH of 4.0, and an enzymatic hydrolysis time of 4 h, and a solid-to-liquid ratio of 1:10, royal jelly protein peptides below 1000 Da were prepared, which have certain hypoglycemic and antioxidant activities.

[0005] The content of melanin in the skin is the main factor determining skin color, and melanin can protect the skin from ultraviolet burns. However, excessive production of melanin can lead to skin diseases such as melasma, freckles, and even melanoma. Currently, many traditional melanin inhibitors such as kojic acid and hydroquinone can cause skin inflammation and have disadvantages such as low skin permeability and instability. Therefore, it is of great significance to develop new, safe, and highly effective natural melanin inhibitors. Summary of the Invention

[0006] Aiming at the current lack of safe and highly effective melanin inhibitors, the present invention provides a safe and effective natural melanin production inhibitor, which is derived from royal jelly, obtained by protease hydrolysis, and has good product safety.

[0007] Another object of the present invention is to provide an application of the above melanin inhibitor in cosmetics or drugs.

[0008] To achieve the above object, the present invention adopts the following technical solutions.

[0009] A preparation method of royal jelly enzymatic hydrolysate includes the following steps:

[0010] (1) Enzymatically hydrolyze the soluble royal jelly protein solution with pepsin to obtain an enzymatic hydrolysate;

[0011] (2) Ultrafilter and separate the enzymolysis solution to obtain a polypeptide solution with a molecular weight less than 3 KDa;

[0012] The concentration of the soluble royal jelly protein solution is 1.5% - 2%, and the addition amount of the protease is 5% - 7% of the soluble royal jelly protein; the enzymolysis time is 4 h - 5 h, and the pH is 2.0 - 3.0. Preferably, the pH is 2.0.

[0013] The preparation method of the soluble royal jelly protein includes the following steps:

[0014] (i) Dissolve royal jelly and then centrifuge to obtain supernatant I;

[0015] (ii) Centrifuge supernatant I again to obtain supernatant II;

[0016] (iii) Lyophilize supernatant II to obtain soluble royal jelly protein.

[0017] In step (i), the material - liquid ratio of royal jelly to the solvent is 1:8 (g / mL). The solvent is selected from water or a phosphate buffer solution with pH 7.0; the concentration of the buffer solution is 50 mM.

[0018] Preferably, in order to dissolve royal jelly protein better and improve the yield of soluble royal jelly protein, in step (i), the solvent is a phosphate buffer solution. Correspondingly, in order to improve the purity of soluble royal jelly protein, before lyophilization in step (iii), a dialysis step may also be included.

[0019] To assist in dissolving royal jelly, ultrasonic assistance can be used. The power of the ultrasonic wave is 180 W, and the time is 40 min.

[0020] In steps (i) and (ii), the centrifugation rate is 10000 - 12000 rpm, the centrifugation time is 20 - 40 min, and the centrifugation temperature is 4°C.

[0021] Preferably, in the above - mentioned preparation method, after step (2), a step of concentrating or drying the polypeptide solution is further included.

[0022] A royal jelly protein hydrolysate obtained by the above - mentioned preparation method.

[0023] The above - mentioned royal jelly protein hydrolysate can be used as a whitening active ingredient and added to drugs or cosmetics.

[0024] A food, health food, cosmetic or drug containing the above - mentioned royal jelly protein hydrolysate.

[0025] The present invention has the following advantages:

[0026] The present invention uses royal jelly as a raw material. By extracting royal jelly protein and using protease enzymatic hydrolysis technology and ultrafiltration separation technology, a low-molecular-weight royal jelly protease hydrolysate (<3KDa) is prepared. Through in vitro experiments, experiments on mouse melanoma cells (B16-F10), and zebrafish experiments, it is proved that the low-molecular-weight royal jelly protease hydrolysate has good melanogenesis inhibitory activity and can reduce the generation of melanin in mouse melanoma cells and zebrafish. This inhibitor comes from the natural food royal jelly, is green and safe, and the preparation process has controllable conditions, is convenient to extract, does not require artificial synthesis, and is expected to be applied to drugs or cosmetics with whitening effects. Description of the Drawings

[0027] Figure 1 Effects of different proteases on tyrosinase inhibitory activity and polypeptide yield;

[0028] Figure 2 Optimization of pepsin enzymatic hydrolysis conditions, where A is different enzyme-substrate ratios; B is different enzymatic hydrolysis times; C is different substrate concentrations; D is different enzymatic hydrolysis pH values;

[0029] Figure 3 Safety evaluation of component III (<3KDa) on B16-F10 cells;

[0030] Figure 4 Effect of component III (<3KDa) on melanogenesis in B16-F10 cells

[0031] Figure 5 Effect of component III (<3KDa) on tyrosinase activity in B16-F10 cells;

[0032] Figure 6 Change in melanin content in zebrafish after 48h of different treatments;

[0033] Figure 7 Determination of melanin content (A) and tyrosinase activity (B) in zebrafish. Detailed Embodiments

[0034] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0035] Example 1 Preparation of Royal Jelly Protease Hydrolysate

[0036] 1. Preparation of Soluble Royal Jelly Protein

[0037] Take the royal jelly sample and place it in a beaker. Add phosphate buffer (50 mM) with pH = 7.0 according to the solid-liquid ratio of 1:8 (g / mL). Then mix it in an ultrasonic cell disruptor (180 W, 40 min) until it is uniformly mixed and does not form layers, and transfer it to a refrigerated centrifuge. Centrifuge at 4°C and a rotation speed of 12,000 r / min for 30 min. After centrifugation, discard the precipitate, and take the supernatant and repeat the above centrifugation once until the resulting solution is clear, transparent, and slightly yellow. Place this solution in a 7KD dialysis bag and dialyze for 24 h to remove small molecular substances in the solution. After dialysis, centrifuge the sample in the dialysis bag for another 30 min to discard the precipitate particles, and put the obtained filtrate into a freeze dryer and freeze-dry for 24 h to obtain freeze-dried royal jelly protein powder.

[0038] 2. Optimization of enzymatic hydrolysis conditions

[0039] (1) Types of proteases

[0040] Dissolve the freeze-dried royal jelly protein powder into a 2.5% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH to the optimal pH of each enzyme, and then add pepsin (pH = 2.0), neutral protease (pH = 7.0), trypsin (pH = 8.0), papain (pH = 7.5), and chymotrypsin (pH = 8.0) respectively until the enzyme-substrate ratio is 1%. Incubate at 37°C, 50°C, 37°C, 55°C, and 37°C for 4 h respectively, and then inactivate the enzyme by boiling water bath for 5 min. Centrifuge the enzymatic hydrolysate at 4°C and 10,000 r / min for 15 min, collect the supernatant, and measure the polypeptide yield and tyrosinase inhibition rate.

[0041] Determination and calculation method of polypeptide yield:

[0042] Take 2.5 mL of the enzymatic hydrolysate, add 2.5 mL of 10% (w / v) trichloroacetic acid (TCA) aqueous solution, mix well on a vortex mixer, let it stand for 10 min, and then centrifuge at 4000 r / min for 15 min. Transfer all the supernatant to a 50 mL volumetric flask and dilute it to the mark with 5% TCA, and shake well. Then take 6.0 mL of the above solution and place it in another test tube, add 4.0 mL of biuret reagent (sample solution: biuret reagent = 3:2, v / v), mix well on a vortex mixer, let it stand for 10 min, and centrifuge at 2000 r / min for 10 min. Measure the OD value of the supernatant at 540 nm. Using bovine serum albumin as a standard, prepare standard solutions of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg / mL with 5% TCA in the same way, measure the OD value and draw a standard curve to obtain the polypeptide concentration (mg / mL) in the sample solution;

[0043] Yield of sample polypeptide (%) = Concentration of polypeptide in enzymatic hydrolysate × Volume of enzymatic hydrolysate / Total amount of raw material × 100.

[0044] Method for determination and calculation of tyrosinase inhibition rate:

[0045] The total volume of the reaction system is 200 μL, and the entire sample addition process is carried out on ice. Add 80 μL of the sample and 40 μL of tyrosinase (375 U / mL) to a 96-well plate, and then incubate at 37 °C for 10 minutes. Subsequently, add 80 μL of L-DOPA (2 mM) to each well and incubate at 37 °C for another 5 minutes. Then, measure the absorbance at 475 nm using a microplate reader. Calculate the tyrosinase inhibition rate:

[0046] Tyrosinase inhibition rate (%) = (1 - ) × 100%

[0047] In the formula: T is the absorbance value of the sample tube, that is, the absorbance value of the solution after the sample reacts with tyrosinase; T 0 is the background absorbance value of the sample; C is the average value of the absorbance values of the enzyme reaction tubes measured 3 times, that is, the absorbance value of the reaction between tyrosinase and dopa without adding the sample; C 0 is the background absorbance value of the solvent.

[0048] The polypeptide yields and tyrosinase inhibition rates of different proteases are as Figure 1 shown: The enzymatic hydrolysates of pepsin, neutral protease, trypsin, papain, and chymotrypsin all have tyrosinase inhibition activity. Among them, the pepsin hydrolysate has the best inhibitory effect on tyrosinase activity and has a relatively high polypeptide yield.

[0049] (2) Enzyme-substrate ratio

[0050] Dissolve the freeze-dried royal jelly protein powder into a 2.5% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH to 2, and then add pepsin to the enzyme-substrate ratio of 1% - 9%; incubate at 37 °C for 4 h, and inactivate the enzyme by boiling water bath for 5 min after completion. Centrifuge the enzymatic hydrolysate at 4 °C and 10,000 r / min for 15 min, collect the supernatant, and measure the polypeptide yield and tyrosinase inhibition rate.

[0051] As can be seen from Figure 2 A, when the enzyme-substrate ratio is 5% - 7%, the inhibitory effect of the pepsin hydrolysate on tyrosinase is the best, significantly higher than other groups ( p <0.05); when the enzyme-substrate ratio is 7% - 9%, the polypeptide yield is significantly higher than other groups ( p <0.05). Considering the enzymatic hydrolysis effect and cost, the optimal enzyme-substrate ratio is 7%.

[0052] (3) Enzymatic hydrolysis time

[0053] Dissolve the freeze-dried royal jelly protein into a 2.5% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH to 2, and then add pepsin to each part until the enzyme-substrate ratio is 7%; carry out enzymatic hydrolysis at a constant temperature of 37 °C for 2 - 6 h, and after completion, inactivate the enzyme by boiling water bath for 5 min. Centrifuge the enzymatic hydrolysate at 4 °C and 10,000 r / min for 15 min, collect the supernatant, and measure the polypeptide yield and tyrosinase inhibition rate.

[0054] As can be seen from Figure 2 B, when the enzymatic hydrolysis time is 4 - 5 h, the inhibition rate of the enzymatic hydrolysate on tyrosinase is significantly higher than that of other groups ( p <0.05); there is no significant difference in the polypeptide yield during enzymatic hydrolysis for 3 - 6 h ( p >0.05). Therefore, the optimal enzymatic hydrolysis time is 4 h.

[0055] (4) Substrate concentration

[0056] Dissolve the freeze-dried royal jelly protein into a 1 - 4% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH to 2, and then add pepsin to each part until the enzyme-substrate ratio is 7%; carry out enzymatic hydrolysis at a constant temperature of 37 °C for 4 h, and after completion, inactivate the enzyme by boiling water bath for 5 min. Centrifuge the enzymatic hydrolysate at 4 °C and 10,000 r / min for 15 min, collect the supernatant, and measure the polypeptide yield and tyrosinase inhibition rate.

[0057] As can be seen from Figure 2 C, when the substrate concentration is 1.5% - 2%, the inhibitory activity of the enzymatic hydrolysate on tyrosinase is significantly higher than that of other concentration groups ( p <0.05); and the change in substrate concentration has a significant impact on the polypeptide yield, and the polypeptide yield is the highest at 1.5%. Therefore, 1.5% is used as the optimal substrate concentration.

[0058] (5) Enzymatic hydrolysis pH

[0059] Dissolve the freeze-dried royal jelly protein into a 1.5% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH value to 2.0 - 4.0 respectively, and then add pepsin to each part until the enzyme-substrate ratio is 7%; carry out enzymatic hydrolysis at a constant temperature of 37 °C for 4 h, and after completion, inactivate the enzyme by boiling water bath for 5 min. Centrifuge the enzymatic hydrolysate at 4 °C and 10,000 r / min for 15 min, collect the supernatant, and measure the polypeptide yield and tyrosinase inhibition rate.

[0060] As can be seen from Figure 2 D, when the enzymatic hydrolysis pH is 2.0, both the inhibitory activity of the enzymatic hydrolysate on tyrosinase and the polypeptide yield are significantly higher than those of other pH groups ( p <0.05).

[0061] From the tyrosinase inhibition rates of the enzymolysates under the above different conditions, it can be seen that the type of protease, the enzyme-substrate ratio, the enzymolysis time, the substrate concentration, and the enzymolysis pH will all lead to different enzymolysis effects, thereby affecting the tyrosinase inhibitory activity of the enzymolysate; insufficient or excessive enzymolysis will have an adverse effect on the inhibitory activity of the enzymolysate.

[0062] 3. Ultrafiltration separation of enzymolysate

[0063] Dissolve the freeze-dried royal jelly protein powder into a 1.5% royal jelly protein solution with ultrapure water, divide it into several equal parts, adjust the pH to 2, and then add pepsin to the enzyme-substrate ratio of 7%; enzymolyze at 37 °C for 4 h, and after completion, inactivate the enzyme by boiling water bath for 5 min to obtain the enzymolysis solution.

[0064] Successively use 10KDa and 3KDa ultrafiltration membranes to ultrafilter and separate the enzymolysis solution according to the molecular weight size to obtain three components: component I (>10KDa), component II (3-10KDa), and component III (<3KDa). After freeze-drying the three components respectively, make royal jelly protease hydrolysis solutions with different concentrations (mg / mL), and evaluate the in vitro tyrosinase inhibitory activity, using the synthetic polypeptide IIPFIF as a control.

[0065] Table 1 Tyrosinase inhibitory activity of enzymolysis products with different molecular weights

[0066]

[0067] The results are shown in Table 1. Component I (>10KDa), component II (3-10KDa), and component III (<3KDa) all have tyrosinase inhibitory activity. Among them, component III (<3KDa) has the strongest inhibitory activity on tyrosinase, and the inhibitory rate on tyrosinase at a concentration of 1-5 mg / mL is higher than that of component I (>10KDa), component II (3-10KDa), and the control. The inhibitory rate of the <3KDa component on tyrosinase reaches 93.0% at a concentration of 5 mg / mL. The IC 50 values of component I (>10KDa), component II (3-10KDa), and component III (<3KDa) are 4.27 mg / mL, 3.33 mg / mL, and 2.88 mg / mL respectively. Therefore, component III (<3KDa) is selected to further verify its activity with B16-F10 cells and zebrafish.

[0068] Example 2 Safety of royal jelly protease hydrolysate

[0069] Take B16-F10 cells in the logarithmic growth phase, passage and digest them with 0.25% trypsin, and prepare a single-cell suspension of 2.5×10 5 / mL with DMEM medium. Inoculate the cells into a 96-well plate, and keep 2.5×10 4 / Inoculation density of the pores, placed in an incubator at 37 °C and 5% CO 2 for incubation. After 12 hours, discard the original culture medium, and add 0.1 mg / mL kojic acid and component III (<3KDa) prepared in Example 1 at different concentrations respectively. The blank group and the control group replace the fresh culture medium. Continue to culture for 24 h. The cell viability was determined by the MTT method.

[0070] The effects of different concentrations of component III (<3KDa) on the viability of B16-F10 cells are as Figure 3 shown. Component III (<3KDa) has no significant effect on cell viability at concentrations of 0.1 mg / mL - 1.5 mg / mL and is safe.

[0071] Example 3 Effect of royal jelly protease hydrolysate treatment on the melanin content of B16-F10 cells

[0072] Inoculate B16-F10 cells in the logarithmic phase into a 96-well plate at a density of 5.0×10 5 / well. After culturing for 12 hours, discard the original culture medium, and add 0.1 mg / mL kojic acid and component III prepared in Example 1 at different concentrations respectively. The blank group and the control group replace the fresh culture medium and continue to culture for 24 h. Discard the supernatant and wash twice with PBS. Add 1 mL of 1M NaOH (containing 10% DMSO) to each well, and place the well plate in an 80 °C oven to dissolve melanin for 2 h. Take out the well plate and let it return to room temperature. Transfer the dissolved cells into a 1.5 mL centrifuge tube and centrifuge at 10000 rpm for 10 min. After centrifugation, take 100 μL of the supernatant and transfer it into a 96-well plate, and measure the absorbance at 405 nm. Calculate the cell melanin content according to the following formula:

[0073] Relative melanin content (%) = [(A - C) / (B - C)] × 100%

[0074] where A is the average absorbance (OD) value of each concentration in the sample group, B is the average absorbance (OD) value of the control group, and C is the average absorbance (OD) value of the blank group.

[0075] From Figure 4 it can be seen that component III (<3KDa) at different concentrations all showed melanin inhibitory activity. After acting for 24 h at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, the relative cell melanin contents were 89.3%, 73.8%, 61.0%, and 54.5% respectively, and the melanin inhibition rates were 10.7%, 26.2%, 39%, and 45.5% respectively.

[0076] Example 4 Effect of royal jelly protease hydrolysate treatment on the tyrosinase activity of B16-F10 cells

[0077] The logarithmic-phase B16-F10 cells were seeded into a 96-well plate at a density of 2.5×10 5 / well, and after culturing for 12 hours, the original culture medium was discarded. Then, 0.1 mg / mL kojic acid and component III prepared in Example 1 at different concentrations were added respectively. The blank group and the control group were replaced with fresh culture medium and continued to be cultured for 24 h. The supernatant was discarded and washed twice with PBS. 0.1 mL of 1% TritonX-100 solution was added to each well, and it was quickly placed at -80 °C and stored for 30 min, then frozen and thawed repeatedly 3 times, and then thawed at room temperature to completely rupture the cells. Then, the cell lysate was centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was taken, and the activity of tyrosinase in B16-F10 cells was measured by the L-DOPA oxidation method.

[0078] From Figure 5 it can be seen that when the concentration of component III (<3 KDa) exceeds 0.1 mg / mL, the inhibitory effect on tyrosinase is significant. After acting for 24 h at 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, the relative tyrosinase activities in the cells are 83.0%, 55.5%, and 41.2% respectively, and the inhibitory rates of tyrosinase activity are 17%, 44.5%, and 58.8% respectively.

[0079] Example 5 Effects of royal jelly protease hydrolysate treatment on the melanin content and tyrosinase activity in zebrafish

[0080] The zebrafish embryos fertilized for 24 h were placed in an incubator at a constant temperature of 28.5 °C for culture. Different concentration treatment groups of royal jelly protease hydrolysate (<3 KDa) (0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL), kojic acid treatment group (0.1 mg / mL) were set up, and the blank control was the embryos cultured in zebrafish embryo culture medium without the test substance. A 96-well plate was used as the culture container, and an appropriate amount of embryo culture medium was added to each well. The morphology of zebrafish embryos in each group was observed and recorded. After the test substance was treated for 48 h, 30 zebrafish were randomly selected from each concentration to measure the tyrosinase activity and melanin production in vivo. All zebrafish treated with component III (<3 KDa) in the concentration range of 0.01 mg / mL - 0.2 mg / mL survived and had normal physiological states.

[0081] After 48 h of treatment with component III (<3 KDa), the zebrafish were placed under a stereomicroscope to observe the morphology and melanin distribution. The results are as Figure 6 shown, where a is the control group, b is the 0.1 mg / mL kojic acid treatment group, and c-f are the component III treatment groups with concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL in sequence. It can be observed from the above pictures that the melanin in the abdominal yolk and on both sides of the back of the zebrafish treated with component III (<3 KDa) was significantly reduced.

[0082] Figure 7 (A) shows the results of melanin content. It shows that when the concentration exceeds 0.01 mg / mL, the inhibitory effect of Component III (<3KDa) on melanin is significant. Under the treatment of Component III (<3KDa) with concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, the melanin content in zebrafish is 86.6%, 74.7%, and 56.5% respectively, which is decreased by 13.4%, 25.3%, and 43.5% respectively compared with the control group.

[0083] From Figure 7 (B), it can be seen that Component III (<3KDa) can significantly reduce the tyrosinase activity in zebrafish. Under the treatment of Component III (<3KDa) with concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, the tyrosinase activities in zebrafish are 73.6%, 69.8%, and 62.9% in sequence. Therefore, Component III (<3KDa) has a good inhibitory effect on melanin in the zebrafish model and can reduce the melanin production by inhibiting the tyrosinase activity.

[0084] In summary, it can be known that the proteolytic product of royal jelly with <3KDa has the effects of inhibiting tyrosinase activity and reducing melanin production both in vitro and in vivo, and can be used as a whitening active ingredient.

[0085] The above description is only for the embodiments of the present application and is not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A method for preparing a royal jelly protein hydrolysate having melanin inhibitory activity, characterized in that: The following steps are involved: (1) hydrolyzing the soluble royal jelly protein solution with pepsin to obtain an enzymatic hydrolyzate; (2) ultrafiltration separation of the enzymatic hydrolysate to obtain a polypeptide solution with a molecular weight less than 3 KDa; The concentration of the soluble royal jelly protein solution is 1.5%-2%, and the amount of protease added is 5%-7% of the soluble royal jelly protein; the enzymatic hydrolysis time is 4h-5h, and the pH is 2.0-3.

0.

2. The preparation method according to claim 1, characterized in that: The pH of the enzymatic hydrolysis was 2.0; After step (2), the method further includes concentrating or drying the polypeptide solution.

3. The preparation method according to claim 1, characterized in that: The method for preparing the soluble royal jelly protein comprises the following steps: (i) dissolving the royal jelly and centrifuging to obtain a supernatant I; (ii) centrifuging the supernatant I again to obtain supernatant II; (iii) The supernatant II is freeze-dried to obtain soluble royal jelly protein.

4. The preparation method according to claim 3, characterized in that: In step (i), the solid-liquid ratio of royal jelly to solvent is 1:8 (g / mL); the solvent is selected from water or phosphate buffer with pH 7.

0.

5. The preparation method according to claim 3, characterized in that: In step (i), the solvent is a phosphate buffer; the concentration of the buffer is 50 mM; in step (iii), a dialysis step is also included before freeze-drying.

6. The preparation method according to claim 3, characterized in that: In step (i), the royal jelly is dissolved by ultrasonic dissolution; the power of the ultrasound is 180W and the time is 40 minutes; In steps (i) and (ii), the centrifugal speed is 10000-12000 rpm, the centrifugal time is 20-40 min, and the centrifugal temperature is 4°C.

7. A royal jelly protein hydrolysate obtained by the preparation method as described in any one of claims 1 to 6.

8. Use of the royal jelly protein hydrolysate according to claim 7 in the preparation of cosmetics or medicines, characterized in that: Royal jelly protein hydrolysate as whitening active ingredient.

9. A food, cosmetic or medicine comprising the royal jelly protein hydrolysate according to claim 7.

10. A health food comprising the royal jelly protein hydrolysate according to claim 7.

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