Royal jelly protein zymolyte with melanin inhibition activity as well as preparation and application of royal jelly protein zymolyte

Abstract

The invention belongs to the technical field of deep processing of agricultural products, and provides royal jelly protein zymolyte with melanin inhibition activity and preparation and application of the royal jelly protein zymolyte. Performing enzymolysis on the soluble royal jelly protein solution with pepsin to obtain enzymatic hydrolysate; and carrying out ultrafiltration separation on the enzymatic hydrolysate to obtain a polypeptide solution with the molecular weight of less than 3KDa, namely royal jelly protein hydrolysate. The royal jelly is used as a raw material, the royal jelly protein hydrolysate with low molecular weight is prepared by extracting royal jelly protein and utilizing a protease enzymolysis technology and an ultrafiltration separation technology, and the royal jelly protein hydrolysate has good melanogenesis inhibition activity and can reduce generation of mouse melanoma cells and zebra fish melanin. The inhibitor is derived from natural food royal jelly, is green and safe, is controllable in conditions in the preparation process, is convenient to extract, does not need artificial synthesis, and is expected to be applied to food, medicines or cosmetics with a whitening effect.

Description

Technical Field

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

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Royal jelly is a pulpy substance secreted by the hypopharyngeal gland and maxillary gland of worker bees. It has biological activities such as immunomodulation, antibacterial, antioxidant, and anti-aging, and is widely used in health care products, cosmetics, and other fields. Royal jelly is rich in protein, and its protein content accounts for about 9-18% of fresh royal jelly and 30%-50% of royal jelly dry matter. It is an important active ingredient in royal jelly and has biological activities such as antioxidant, antibacterial, anti-inflammatory, immunomodulatory, wound healing, and anti-aging. Although royal jelly protein has significant biological activity, it has a large molecular weight, is not easily absorbed and utilized by human skin, and has the disadvantages of being easy to cause allergies. Therefore, increasing the absorption rate and efficacy of royal jelly protein and reducing allergic reactions are of great significance to the development of its products. Proteolysis technology can decompose proteins into small molecular peptides, which have the characteristics of high safety, strong biological activity, easy absorption, and high targeting specificity compared with the original protein, and can effectively improve the biological activity of royal jelly protein and product added value.

[0004] Due to the difference in preparation process, the enzymatic hydrolysate of royal jelly protein will obtain small molecule peptides with different functions. For example, CN113647591 A discloses a royal jelly enzymatic hydrolysate, which is obtained by enzymatic hydrolysis of royal jelly by protease M-P001; the protease M-P001 is derived from Mycobacterium tuberculosis ( Mycobacterium tuberculosis ). The enzymatic hydrolysis conditions are: using protease M-P001 to enzymatically hydrolyze the royal jelly aqueous solution, the royal jelly concentration in the royal jelly aqueous solution is 5-20%; adding protease M-P001 at an addition amount of 1500-2500U / g (based on royal jelly raw material), pH 6.0-8.0, and enzymatic hydrolysis at 33-36°C for 20-25 hours. The royal jelly enzymatic hydrolysate can significantly improve the clearance ability of mouse monocytes and macrophages, improve the activity of mouse NK cells, and has the effect of improving immune activity. CN 115806588 A discloses a small molecule peptide, whose amino acid sequence is IIPFIF (SEQ IDNO: 1), which is obtained by enzymatic hydrolysis of royal jelly using acidic protease. The small molecule peptide has tyrosinase inhibitory activity, and the IC 50It is 6.16mg / mL. CN 116064708 A discloses a royal jelly polypeptide and three oligopeptides, FDRIW (SEQ ID NO: 2), YPDWSW (SEQ ID NO: 3), and WHDKIF (SEQ ID NO: 4), all of which have the function of inhibiting oxidative stress. The royal jelly solution is reacted with an acidic protease at 30-70°C for 3-8 hours to obtain an enzymatic hydrolyzate; the enzymatic hydrolyzate is separated and eluted by a G-15 dextran gel chromatography column, and a total of 5 elution peaks appear. Any one or more eluates from F1-F5 are collected and freeze-dried to obtain the royal jelly polypeptide. Qian Jiale (Study on the Anti-photoaging Activity of Royal Jelly and Its Enzymatic Hydrolyzates [D]. Zhejiang University, 2021) obtained the main royal jelly protein by dialysis, and the royal jelly and royal jelly main protein were enzymatically hydrolyzed by trypsin and pepsin. The sample was freeze-dried to prepare a freeze-dried powder, and the above royal jelly enzymatic hydrolyzate was detected by the photoaging model of human fibroblasts to show certain anti-photoaging activity. In the study, Zhu Zuoyi et al. (Zhu Zuoyi, Zhang Yu, Wang Junhong, et al. Preparation of royal jelly protein peptides and study of their hypoglycemic and antioxidant activities [J]. Food Industry Science and Technology, 2020, 41(17):7) used acid protease as the enzyme preparation, the enzyme addition amount was 6000U / g, the enzymatic hydrolysis temperature was 43℃, the enzymatic hydrolysis pH was 4.0, the enzymatic hydrolysis time was 4h, and the solid-liquid ratio was 1:10. Under these conditions, royal jelly protein peptides below 1000Da were prepared, which had certain hypoglycemic and antioxidant activities.

[0005] The melanin content in the skin is the main factor that determines skin color. Melanin can protect the skin from ultraviolet burns. However, excessive melanin production can lead to skin diseases such as chloasma, 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 efficient natural melanin inhibitors. Summary of the invention

[0006] In view of the current problem of lack of safe and efficient melanin inhibitors, the present invention provides a safe and effective natural melanin production inhibitor, which is derived from royal jelly and 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, food or medicine.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A method for preparing royal jelly protein hydrolysate comprises the following steps: (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%, 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. Preferably, the pH is 2.0.

[0010] 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.

[0011] In step (i), the solid-liquid ratio of royal jelly to solvent is 1:8 (g / mL). The solvent is selected from water or a phosphate buffer with a pH of 7.0; the concentration of the buffer is 50 mM.

[0012] Preferably, in order to make the royal jelly protein more soluble and improve the yield of soluble royal jelly protein, in step (i), the solvent is phosphate buffer. Accordingly, in order to improve the purity of soluble royal jelly protein, in step (iii), a dialysis step may be included before freeze drying.

[0013] In order to assist the dissolution of royal jelly, ultrasonic dissolution can be used with an ultrasonic power of 180W and a time of 40 minutes.

[0014] In steps (ii) and (iii), the centrifugation speed is 10000-12000 rpm, the centrifugation time is 20-40 min, and the centrifugation temperature is 4°C.

[0015] Preferably, in the above preparation method, step (2) further includes the step of concentrating or drying the polypeptide solution.

[0016] A royal jelly protein hydrolysate obtained by the above preparation method.

[0017] The royal jelly protein hydrolysate can be used as a whitening active ingredient and added to food, health food, medicine or cosmetics.

[0018] A food, health food, cosmetic or medicine containing the royal jelly protein hydrolysate.

[0019] The present invention has the following advantages: The present invention uses royal jelly as raw material, extracts royal jelly protein, and uses protease enzymatic process and ultrafiltration separation technology to prepare low molecular weight royal jelly protein hydrolysate (<3KDa), and proves through in vitro experiments, mouse melanoma cell (B16-F10) experiments and zebrafish experiments that low molecular weight royal jelly protein hydrolysate has good melanin production inhibitory activity, and can reduce the production of mouse melanoma cells and zebrafish melanin. The inhibitor comes from natural food royal jelly, is green and safe, and the preparation process conditions are controllable, the extraction is convenient, and no artificial synthesis is required. It is expected to be used in foods, drugs or cosmetics with whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effects of different proteases on tyrosinase inhibitory activity and peptide yield; Figure 2 The conditions for pepsin hydrolysis are optimized, where A is different enzyme-substrate ratios; B is different hydrolysis times; C is different substrate concentrations; and D is different hydrolysis pH values. Figure 3 To evaluate the safety of component III (<3KDa) on B16-F10 cells; Figure 4 Effect of component III (<3KDa) on melanin production in B16-F10 cells Figure 5 The effect of component III (<3KDa) on tyrosinase activity of B16-F10 cells; Figure 6 The changes of melanin content in zebrafish after 48 hours of different treatments; a is the control group, b is the 0.1 mg / mL kojic acid treatment group, and cf are the concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL component III treatment groups, respectively; Figure 7 Determination of melanin content (A) and tyrosinase activity (B) in zebrafish. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Preparation of Royal Jelly Protein Hydrolysate 1. Preparation of soluble royal jelly protein Take a royal jelly sample and place it in a beaker, add phosphate buffer (50mM) with pH=7.0 at a solid-liquid ratio of 1:8 (g / mL), then transfer it to a refrigerated centrifuge after mixing it in an ultrasonic cell crusher (180W, 40min) until it is uniform and not stratified, and then centrifuge it at 4°C and a speed of 12000r / min for 30min; after centrifugation, discard the precipitate, take the supernatant and repeat the above centrifugation once until the obtained solution is clear, transparent and slightly yellow; place the solution in a 7KD dialysis bag and dialyze it for 24h to remove small molecules in the solution; after dialysis, centrifuge the sample in the dialysis bag again for 30min to discard the precipitate particles, and place the obtained filtrate in a freeze dryer and freeze-dry for 24h to obtain royal jelly protein freeze-dried powder.

[0023] 2. Optimization of enzymatic hydrolysis conditions (1) Types of proteases Dissolve the lyophilized royal jelly protein powder in ultrapure water to form a 2.5% royal jelly protein solution, divide it into several portions, adjust the pH to the optimum 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) to an enzyme-substrate ratio of 1%; perform enzymolysis at 37℃, 50℃, 37℃, 55℃ and 37℃ for 4h, and then inactivate the enzyme in a boiling water bath for 5min. Centrifuge the enzymolysis solution at 4℃ and 10000 r / min for 15min, collect the supernatant, and determine the peptide yield and tyrosinase inhibition rate.

[0024] Determination and calculation method of peptide yield: Take 2.5mL of enzymatic hydrolysate, add 2.5mL of 10% (w / v) trichloroacetic acid (TCA) aqueous solution, mix evenly on a vortex mixer, let stand for 10min, then centrifuge at 4000r / min for 15min, transfer all the supernatant to a 50mL volumetric flask, and dilute to the mark with 5% TCA, shake well. Then take 6.0mL of the above solution and place it in another test tube, add 4.0mL of biuret reagent (sample solution: biuret reagent = 3:2, v / v), mix evenly on a vortex mixer, let stand for 10min, and centrifuge at 2000r / min for 10min. Take the supernatant and measure the OD value at 540 nm. Using bovine serum albumin as the standard, 5% TCA was used to prepare standard solutions of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 mg / mL, and the OD values ​​were determined by the same method and the standard curve was drawn to obtain the polypeptide concentration (mg / mL) in the sample solution; Sample peptide yield (%) = peptide concentration in hydrolyzate × volume of hydrolyzate / total amount of raw materials × 100.

[0025] Determination and calculation method of tyrosinase inhibition rate: The total volume of the reaction system was 200 μL, and the entire sample addition process was performed on ice. 80 μL of sample and 40 μL of tyrosinase (375 U / mL) were added to a 96-well plate and then incubated at 37 ° C for 10 minutes. Subsequently, 80 μL of L-DOPA (2 mM) was added to each well and incubated at 37 ° C for another 5 minutes. Then, the absorbance was measured at 475 nm using a microplate reader. Calculate the tyrosinase inhibition rate: Tyrosinase inhibition rate (%) = (1- )×100% In the formula: T is the absorbance of the sample tube, that is, the absorbance of the solution after the sample reacts with tyrosinase; T0 is the background absorbance of the sample; C is the average of the three absorbances of the enzyme reaction tube, that is, the absorbance of the reaction of tyrosinase and DOPA when no sample is added; C0 is the background absorbance of the solvent.

[0026] The peptide yields of different proteases and the inhibition rates of tyrosinase are shown in Figure 2. Figure 1 As shown: the hydrolysates of pepsin, neutral protease, trypsin, papain and chymosin all have tyrosinase inhibitory activity, among which the pepsin hydrolysate has the best inhibitory effect on tyrosinase activity and has a higher polypeptide yield.

[0027] (2) Enzyme-substrate ratio Dissolve the freeze-dried royal jelly protein powder in ultrapure water to form a 2.5% royal jelly protein solution, divide it into several portions, adjust the pH to 2, and then add pepsin to a base ratio of 1%-9%; perform constant temperature enzymolysis at 37°C for 4 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes. Centrifuge the enzymolysis solution at 4°C and 10,000 r / min for 15 minutes, collect the supernatant, and determine the peptide yield and tyrosinase inhibition rate.

[0028] Depend on Figure 2 As shown in Figure A, when the enzyme-substrate ratio was 5%-7%, the inhibitory effect of pepsin hydrolysate on tyrosinase was the best, significantly higher than that of other groups ( p <0.05); when the enzyme-substrate ratio was 7%-9%, the peptide yield was significantly higher than that of other groups ( p <0.05). Considering the enzymatic hydrolysis effect and cost, 7% enzyme-substrate ratio is the optimal enzyme-substrate ratio.

[0029] (3) Enzymatic hydrolysis time Dissolve the freeze-dried royal jelly protein powder in ultrapure water to form a 2.5% royal jelly protein solution, divide it into several portions, adjust the pH to 2, and then add pepsin to a 7% enzyme-substrate ratio; perform constant temperature enzymolysis at 37°C for 2-6 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes. Centrifuge the enzymolysis solution at 4°C and 10,000 r / min for 15 minutes, collect the supernatant, and determine the peptide yield and tyrosinase inhibition rate.

[0030] Depend on Figure 2 As shown in Figure B, when the enzymatic hydrolysis time was 4-5h, the inhibition rate of tyrosinase by the enzymatic hydrolysate was significantly higher than that of other groups ( p <0.05); the difference in peptide yield was not significant between 3 and 6 h of enzymatic hydrolysis ( p >0.05). Therefore, 4 h of enzymatic hydrolysis was the optimal enzymatic hydrolysis time.

[0031] (4) Substrate concentration Dissolve the royal jelly protein freeze-dried powder in ultrapure water to form a 1-4% royal jelly protein solution, divide it into several portions, adjust the pH to 2, and then add pepsin to a 7% enzyme-substrate ratio; perform constant temperature enzymolysis at 37°C for 4 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes. Centrifuge the enzymolysis solution at 4°C and 10,000 r / min for 15 minutes, collect the supernatant, and determine the peptide yield and tyrosinase inhibition rate.

[0032] Depend on Figure 2 As shown in Figure C, when the substrate concentration was 1.5%-2%, the inhibitory activity of the hydrolysate on tyrosinase was significantly higher than that of other concentration groups ( p <0.05); and the change of substrate concentration has a significant effect on the peptide yield, and the peptide yield is the highest at 1.5%. Therefore, 1.5% is taken as the optimal substrate concentration.

[0033] (5) Enzymatic hydrolysis pH Dissolve the freeze-dried royal jelly protein powder in ultrapure water to form a 1.5% royal jelly protein solution, divide it into several portions, adjust the pH value to 2.0-4.0, and then add pepsin to a 7% enzyme-substrate ratio; perform constant temperature enzymolysis at 37°C for 4 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes. Centrifuge the enzymolysis solution at 4°C and 10,000 r / min for 15 minutes, collect the supernatant, and determine the peptide yield and tyrosinase inhibition rate.

[0034] Depend on Figure 2 As shown in D, when the enzymatic hydrolysis pH was 2.0, the inhibitory activity of the enzymatic hydrolysate on tyrosinase and the yield of peptides were significantly higher than those in other pH groups ( p <0.05).

[0035] From the tyrosinase inhibition rate of the hydrolysates under the above different conditions, it can be seen that the type of protease, enzyme-substrate ratio, hydrolysis time, substrate concentration, and hydrolysis pH will lead to different enzymatic cleavage effects, thereby affecting the tyrosinase inhibitory activity of the hydrolysates; insufficient or excessive hydrolysis will have an adverse effect on the inhibitory activity of the hydrolysates.

[0036] 3. Ultrafiltration separation of enzymatic hydrolysate Dissolve the royal jelly protein freeze-dried powder with ultrapure water to form a 1.5% royal jelly protein solution, divide it into several portions, adjust the pH to 2, and then add pepsin to a 7% enzyme-substrate ratio; perform constant temperature enzymolysis at 37°C for 4 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes to obtain the enzymolysis solution.

[0037] The enzymatic hydrolysate was separated by ultrafiltration using 10KDa and 3KDa ultrafiltration membranes according to molecular weight to obtain three components: component I (>10KDa), component II (3-10KDa) and component III (<3KDa). The three components were freeze-dried and prepared into royal jelly enzymatic hydrolysate solutions of different concentrations (mg / mL) for in vitro tyrosinase inhibitory activity evaluation, with the synthetic peptide IIPFIF (SEQ ID NO: 1) as the control.

[0038] Table 1 Tyrosinase inhibitory activity of hydrolysates with different molecular weights 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 against tyrosinase. The inhibition rate of tyrosinase at a concentration of 1-5mg / mL is higher than that of component I (>10KDa), component II (3-10KDa) and the control. The inhibition rate of the <3KDa component against tyrosinase at a concentration of 5mg / mL reaches 93.0%. The IC values ​​of component I (>10KDa), component II (3-10KDa) and component III (<3KDa) are as follows: 50 The results were 4.27 mg / mL, 3.33 mg / mL, and 2.88 mg / mL, respectively. Therefore, component III (<3 KDa) was selected to further verify its activity using B16-F10 cells and zebrafish.

[0039] Example 2 Safety of Royal Jelly Protein Hydrolysate B16-F10 cells grown to the logarithmic phase were taken and digested with 0.25% trypsin, and the cells were diluted to 2.5×10 5 / mL single cell suspension, and cells were seeded into 96-well plates at a constant rate of 2.5×10 4 / well, and cultured in a constant temperature incubator at 37°C and 5% CO2. After 12 hours, the original culture medium was discarded, and 0.1 mg / mL kojic acid and different concentrations of component III (<3 KDa) prepared in Example 1 were added, and fresh culture medium was replaced for the blank group and the control group. Culture was continued for 24 hours. Cell viability was determined by MTT method.

[0040] Effects of different concentrations of component III (<3 KDa) on B16-F10 cell viability Figure 3As shown, component III (<3KDa) had no significant effect on cell viability at a concentration of 0.1mg / mL-1.5mg / mL and was safe.

[0041] Example 3 Effect of royal jelly protein hydrolysate treatment on melanin content in B16-F10 cells B16-F10 cells in the logarithmic phase were cultured at 5.0×10 5 / well was inoculated into a 96-well plate. After culturing for 12 hours, the original culture medium was discarded, and 0.1 mg / mL kojic acid and different concentrations of component III prepared in Example 1 were added respectively. The blank group and the control group were replaced with fresh culture medium and cultured for another 24 hours. The supernatant was discarded and washed twice with PBS. 1 mL of 1M NaOH (containing 10% DMSO) was added to each well, and the well plate was placed in an oven at 80°C to dissolve the melanin for 2 hours. Take out the well plate and return it to room temperature. The dissolved cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 rpm for 10 minutes. After centrifugation, 100 μL of supernatant was transferred to a 96-well plate, the absorbance at 405 nm was detected, and the cell melanin content was calculated according to the following formula: Melanin relative content (%) = [(AC) / (BC)]×100% Among them, 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.

[0042] from Figure 4 It can be seen that different concentrations of component III (<3KDa) all showed melanin inhibitory activity. After 24 hours of action at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, the relative content of cellular melanin was 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.

[0043] Example 4 Effect of royal jelly protein hydrolysate treatment on tyrosinase activity in B16-F10 cells B16-F10 cells in the logarithmic phase were cultured at 2.5×10 5 / well were inoculated into 96-well plates. After culturing for 12 hours, the original culture medium was discarded, and 0.1 mg / mL kojic acid and different concentrations of component III prepared in Example 1 were added respectively. The blank group and the control group were replaced with fresh culture medium and cultured for 24 hours. The supernatant was discarded and washed twice with PBS. 0.1 mL of 1% TritonX-100 solution was added to each well, and the cells were quickly frozen at -80°C for 30 minutes, and frozen and thawed three times, and then thawed at room temperature to completely rupture the cells, and then the cell lysate was centrifuged at 12000 rpm at 4°C for 30 minutes. The supernatant was taken and the activity of tyrosinase in B16-F10 cells was determined by L-DOPA oxidation method.

[0044] 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 24 hours of action at 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, the relative tyrosinase activity in the cells was 83.0%, 55.5%, and 41.2%, respectively, and the tyrosinase activity inhibition rates were 17%, 44.5%, and 58.8%, respectively.

[0045] Example 5 Effects of royal jelly protein hydrolysate treatment on melanin content and tyrosinase activity in zebrafish The zebrafish embryos 24 hours after fertilization were cultured in a 28.5℃ constant temperature incubator. Different concentrations of royal jelly protease hydrolysate (<3KDa) treatment groups (0.01mg / mL, 0.05mg / mL, 0.1mg / mL and 0.2mg / mL) and kojic acid treatment groups (0.1mg / mL) were set up, and the blank control was embryos cultured in zebrafish embryo culture medium without the test substance. A 96-well plate was used as a 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 48 hours of treatment with the test substance, 30 zebrafish were randomly selected at each concentration to determine the tyrosinase activity and melanin production in vivo. All zebrafish treated with component III (<3KDa) in the concentration range of 0.01mg / mL-0.2mg / mL survived and were in normal physiological state.

[0046] After 48 hours of exposure to component III (<3KDa), the zebrafish were placed under a stereomicroscope to observe the morphology and melanin distribution. Figure 6 As shown, a is the control group, b is the 0.1 mg / mL kojic acid treatment group, and cf are the concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL component III treatment groups. It can be observed from the above pictures that the melanin in the abdominal yolk and both sides of the dorsal spine of zebrafish treated with component III (<3KDa) is significantly reduced.

[0047] Figure 7(A) shows the melanin content. The results show that when the concentration exceeds 0.01 mg / mL, component III (<3KDa) has a significant inhibitory effect on melanin. When treated with component III (<3KDa) at concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, the melanin content in zebrafish was 86.6%, 74.7%, and 56.5%, respectively, which were decreased by 13.4%, 25.3%, and 43.5% compared with the control group.

[0048] from Figure 7 As shown in (B), component III (<3KDa) can significantly reduce the activity of tyrosinase in zebrafish. When treated with component III (<3KDa) at concentrations of 0.05mg / mL, 0.1mg / mL, and 0.2mg / mL, the activity of tyrosinase in zebrafish was 73.6%, 69.8%, and 62.9%, respectively. Therefore, component III (<3KDa) has a good melanin inhibitory effect on the zebrafish model and can reduce the amount of melanin production by inhibiting tyrosinase activity.

[0049] In summary, royal jelly protein hydrolysate of less than 3KDa has the effect of inhibiting tyrosinase activity and reducing melanin production both in vitro and in vivo, and can be used as a whitening active ingredient.

[0050] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing royal jelly protein hydrolysate, 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 (ii) and (iii), the centrifugation speed is 10000-12000 rpm, the centrifugation time is 20-40 min, and the centrifugation 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 preparing food, health food, cosmetics or medicines, characterized in that: Royal jelly protein hydrolysate as whitening active ingredient.

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

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