A mulberry leaf active polypeptide and its preparation method

Abstract

This invention relates to the field of deep processing technology of mulberry leaves, and particularly to an active polypeptide from mulberry leaves and its preparation method. The preparation method involves first pulverizing mulberry leaves and soaking them in water, then pretreating them with aminopeptidase and homogenizing them. Next, PC10F neutral protease and 3.350 acidic protease are added sequentially for two hydrolysations. Finally, the mulberry leaf hydrolysate obtained after the two hydrolysations is centrifuged and passed through a 1000 Da ultrafiltration membrane to obtain the mulberry leaf polypeptide with a molecular weight of less than 1000 Da. The mulberry leaf polypeptide obtained by sequential enzymatic hydrolysis with PC10F neutral protease and 3.350 acidic protease of this invention exhibits significant inhibition of tyrosinase activity within a specific molecular weight range. At a polypeptide content of 0.5%–2.0%, the tyrosinase inhibition rate can reach 20.6%–76.71%. Comparative analysis shows that the inhibition rate of this active peptide against tyrosinase is far higher than that of existing mulberry leaf polyphenol and flavonoid aqueous extracts, and it can play a cosmetic role by inhibiting melanin production.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of mulberry leaves, and particularly to an active polypeptide from mulberry leaves and its preparation method. Background Technology

[0002] Mulberry leaves are a food or traditional Chinese medicine that can be used for both medicinal and culinary purposes. Currently, processed mulberry leaf foods on the Chinese market include mulberry leaf tea, mulberry leaf husk oligosaccharide compressed candies, and mulberry sprout vegetables. However, deep-processed mulberry leaf products are not common, possibly due to the following reasons: firstly, mulberry leaves are prone to browning during processing; secondly, mulberry leaves have a low protein content, making them difficult to extract.

[0003] Currently, there are few research reports and patents related to mulberry leaf peptides. For example, the article "Preparation, Structural Identification and Immunological Activity Analysis of Mulberry Leaf Antioxidant Peptides by Enzymatic Method" reports the use of various proteases to hydrolyze mulberry leaves to obtain a peptide solution that scavenge DPPH free radicals. Another example is a process disclosed in patent application CN202111400110.3 to improve the blood sugar-lowering effect of mulberry leaf peptides, which uses alkaline protease hydrolysis to obtain a blood sugar-lowering peptide. Yet another example is a method for preparing mulberry leaf antioxidant peptides disclosed in patent application CN202111575845.X, which mentions the use of an alkaline extraction and acid precipitation method to extract an antioxidant peptide.

[0004] Regarding the functional studies of mulberry leaf extract, Liu Yinghua et al. mentioned in their article "Inhibitory Effect of Mulberry Leaf Extract on Tyrosinase Activity" that flavonoids obtained through alcohol extraction possess tyrosinase activity. Feng Ganyi et al., in their article "Extraction and In Vitro Antioxidant Determination of Mulberry Leaf Polyphenols," mentioned obtaining an antioxidant substance from the polyphenols contained in the extract.

[0005] No reports have been found in existing literature and patents regarding the use of mulberry leaf extracts to inhibit tyrosinase peptides. Summary of the Invention

[0006] To prepare mulberry leaf active polypeptides, this invention provides a method for preparing mulberry leaf active polypeptides. The method involves first pulverizing mulberry leaves and soaking them in water, then pretreating them with aminopeptidase and homogenizing them. Next, PC10F neutral protease and 3.350 acidic protease are added sequentially for two hydrolysis processes. Finally, the mulberry leaf hydrolysate obtained after the two hydrolysis processes is centrifuged and passed through a 1000 Da ultrafiltration membrane to obtain the mulberry leaf polypeptides with a molecular weight of less than 1000 Da. It should be noted that the PC10F neutral protease used in this invention can be the protease Geobacillus stearothermophilus produced by Amano Amano Enzyme Preparations Trading Co., Ltd., but is not limited to this. The 3.350 acidic protease is produced by Aspergillus niger strain.

[0007] In one embodiment, the mulberry leaves and water are mixed and soaked at a mass ratio of 1:6 to 9, and the soaking time is 30 to 90 minutes.

[0008] In one embodiment, the amount of aminopeptidase added is 0.05% to 0.5% of the mass of the feed solution, and the treatment conditions of the aminopeptidase are pH value to 5.0 to 6.5 and hydrolysis at 40 to 55°C for 30 to 50 minutes.

[0009] In one embodiment, the pretreated solution obtained after aminopeptidase treatment needs to be heated to 70-90°C and kept at that temperature for 0.5-3 hours before homogenization for 1-2 minutes.

[0010] In one embodiment, the amount of PC10F neutral protease added is 3000-7000 U / g, calculated based on the protein content of the raw material.

[0011] In one embodiment, the hydrolysis conditions of the PC10F neutral protease are constant temperature hydrolysis at pH 7.0–8.5 and 50–65°C for 50–100 min.

[0012] In one embodiment, the amount of 3,350 acidic protease added is 3,000 to 7,000 U / g, calculated based on the protein content of the raw materials.

[0013] In one embodiment, the hydrolysis conditions of the 3.350 acidic protease are constant temperature hydrolysis at pH 2.5-3.5 and 40-50°C for 40-80 min.

[0014] In one embodiment, the hydrolysate obtained after hydrolysis of the PC10F neutral protease needs to be heated to 90°C for 8-10 minutes to inactivate the enzyme;

[0015] The hydrolysate obtained after hydrolysis of the 3.350 acidic protease needs to be heated to 90°C for 3-5 minutes to inactivate the enzyme.

[0016] In one embodiment, the mulberry leaf polypeptide is further dried into powder using a spray dryer. Testing showed that when the mulberry leaf polypeptide is dried into powder and diluted to a polypeptide content of 0.5%–2.0%, its tyrosinase inhibition rate can reach 20.6%–76.71%, demonstrating a significant inhibitory effect on tyrosinase.

[0017] In one embodiment, the mulberry leaf hydrolysate that has undergone two peptidase hydrolysis processes is centrifuged at 4000-6000 rpm for 5-10 min before ultrafiltration.

[0018] The present invention also provides a mulberry leaf active polypeptide, which is prepared by any of the above-described methods for preparing mulberry leaf active polypeptides.

[0019] Based on the above, compared with the prior art, the method for preparing mulberry leaf active polypeptides provided by the present invention has the following beneficial effects:

[0020] (1) The mulberry leaf polypeptide obtained by sequential enzymatic hydrolysis of PC10F neutral protease and 3.350 acidic protease in this invention has a significant inhibitory effect on tyrosinase activity within a specific molecular weight range. The tyrosinase inhibition rate can reach 20.6% to 76.71% when the polypeptide content is 0.5% to 2.0%. By comparison, it was found that the inhibition rate of this active peptide on tyrosinase is much higher than that of existing mulberry leaf polyphenol and flavonoid aqueous extracts. It can play a cosmetic role by inhibiting the production of melanin.

[0021] (2) The present invention uses aminopeptidase to pretreat mulberry leaves, which can destroy the binding of leaf protein and fibrous substances in mulberry leaves, thereby increasing the yield of mulberry leaf polypeptides.

[0022] (3) The mulberry leaf polypeptide prepared in this invention not only showed anti-digestion properties after being digested in vitro by simulated gastrointestinal fluid, but also enhanced the inhibition of tyrosinase. Therefore, the mulberry leaf polypeptide prepared in this invention has the potential to be made into oral products, so that its whitening effect can be better exerted.

[0023] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects of the invention and other beneficial effects may be realized and obtained by means of the structures and / or components pointed out in the description and claims. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0026] This invention also provides an operational example of a method for preparing mulberry leaf active polypeptides, the specific steps of which are as follows:

[0027] S100. Raw material pretreatment: After crushing the mulberry leaves, add them to pure water at 65-75℃ at a mass ratio of 1:6-9 and soak for 30-90 minutes.

[0028] S200, aminopeptidase pretreatment: Based on the mass of the soaking solution obtained in step S100, add 0.05% to 0.5% aminopeptidase, adjust the pH value to 5.0 to 6.5 with hydrochloric acid, and hydrolyze at 40 to 55°C for 30 to 50 minutes; then heat to 70 to 90°C and keep warm for 0.5 to 3 hours, and pour into a colloid mill while hot to homogenize for 1 to 2 minutes to obtain a homogenized solution.

[0029] S300, First hydrolysis: Calculate based on the protein content of the raw material, add 3000-7000 U / g of PC10F neutral protease to the homogenized solution, adjust the pH value to 7.0-8.5 with sodium hydroxide solution, hydrolyze at a constant temperature of 50-65℃ for 50-100 min, and after hydrolysis, heat to 90℃ for 8-10 minutes to inactivate the enzyme to obtain the first hydrolysate.

[0030] S400, Second hydrolysis: Based on the protein content of the raw material, after cooling the first hydrolysate obtained in step S300, add 3000-7000 U / g of acidic protease (3.350), adjust the pH to 2.5-3.5 with hydrochloric acid, and hydrolyze at 40-50℃ for 40-80 min. After hydrolysis, heat to 90℃ for 3-5 min to inactivate the enzyme and obtain the second hydrolysate.

[0031] S500, Impurity Removal and Membrane Separation: The second hydrolysate obtained in step S400 is centrifuged at 4000-6000 rpm for 5-10 min, and then passed through a 1000 Da ultrafiltration membrane to obtain mulberry leaf polypeptides with a molecular weight of less than 1000 Da, which are then spray-dried into powder for storage.

[0032] The present invention provides the following embodiments.

[0033] Example 1

[0034] S110. Raw material pretreatment: Take 100g of crushed mulberry leaf powder (protein content: 17.64%), add 900ml of purified water at 65℃, and soak for 30min.

[0035] S120, aminopeptidase pretreatment: Add 1g of aminopeptidase to the above soaking solution, adjust the pH value to 5.0 with hydrochloric acid, and hydrolyze at 40℃ for 30min; then heat to 80℃ and keep warm for 2 hours, and pour into a colloid mill while hot to homogenize for 1min to obtain a homogenized solution.

[0036] S130, First hydrolysis: Take the homogenate, add 52,920 U of PC10F neutral protease (Geobacillus stearothermophilus, Amano Amano Enzyme Preparation Trading Co., Ltd.) according to the protein content of the raw material, adjust the pH value to 7 with sodium hydroxide, and hydrolyze at a constant temperature of 50℃ for 50 min; after hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme, and obtain the first hydrolysate.

[0037] S140, Second hydrolysis: After cooling the first hydrolysate obtained in step S130, add 70,560 U of 3,350 acidic protease (produced by Aspergillus niger strain) according to the protein content of the raw material, adjust the pH to 2.5 with hydrochloric acid, keep at 40℃ for 40 min, and heat to 90℃ to inactivate the enzyme for 3 min to obtain the second hydrolysate.

[0038] S150. Impurity removal and membrane separation: The second hydrolysate obtained in step S140 is centrifuged at 4000 rpm for 5 min, and then ultrafiltered through a 1000 Da ultrafiltration membrane to obtain mulberry leaf polypeptides with a molecular weight of less than 1000 Da, which are then spray-dried into powder for storage.

[0039] The tests showed that the peptide powder prepared in Example 1 inhibited tyrosinase by 20.6% when diluted to 0.5% peptide content, 66.47% when diluted to 1.5% peptide content, and 76.71% when diluted to 2% peptide content.

[0040] It should be noted that the test method for the inhibition rate of tyrosinase in this invention is T / SHRH015~2018 Cosmetics~Tyrosinase Activity Inhibition Test.

[0041] To better illustrate that the preparation method provided by the present invention can obtain mulberry leaf polypeptides with high tyrosinase inhibitory activity, the present invention also provides the following comparative examples 1 to 5:

[0042] Comparative Example 1

[0043] The only difference from Example 1 is that the soaking solution obtained in step S100 was directly heated to 90°C and kept at that temperature for 2 hours without undergoing pretreatment with aminopeptidase. All other operations and process conditions are the same as in Example 1.

[0044] Comparative Example 2

[0045] The only difference from Example 1 is that the PC10F neutral protease is replaced with an enzyme such as NY50C neutral protease (produced by Bacillus amyloliquefaciens, Amano Enzyme Trading Co., Ltd.), and the hydrolysis conditions of NY50C neutral protease are 50°C and pH 7.0. All other operations and process conditions are the same as in Example 1.

[0046] Comparative Example 3

[0047] The only difference from Example 1 is that PC10F neutral protease is replaced with enzymes such as AX neutral protease (Amano Amano Enzyme Preparation Trading Co., Ltd., produced by Aspergillus oryzae), and the hydrolysis conditions of AX protease are 50°C and pH 7.0. All other operations and process conditions are the same as in Example 1.

[0048] Comparative Example 4

[0049] The only difference from Example 1 is that 3.4310 acidic protease (produced by Aspergillus niger strain) is replaced with enzymes such as 3.4310 acidic protease (produced by Aspergillus niger strain), and the hydrolysis conditions of 3.4310 acidic protease are 45°C and pH 2.9. All other operations and process conditions are the same as in Example 1.

[0050] Comparative Example 5

[0051] The only difference from Example 1 is that 537 acidic protease (produced by Aspergillus yusa) is used instead of 3.350 acidic protease (produced by Aspergillus niger), and the hydrolysis conditions of 537 acidic protease are 45°C and pH 3.0. All other operations and process conditions are the same as in Example 1.

[0052] The peptide samples obtained above were prepared into solutions with a peptide concentration of 15 mg / ml, and their tyrosinase inhibition rate was detected. The test results are shown in Table 1.

[0053] Table 1

[0054]

[0055] Note: - Not detected

[0056] As can be seen from the test results in Table 1, the tyrosinase inhibition rate of Comparative Example 1, which was not pretreated with aminopeptidase, was 27.17% lower than that of Example 1. The significant decrease in inhibition rate may be due to the fact that aminopeptidase is an exonuclease, and the untreated mulberry leaves do not open the polypeptide chains, which is not conducive to subsequent hydrolysis.

[0057] Comparative Examples 2 and 3 showed no inhibitory effect on tyrosinase when the PC10F neutral protease required for the first hydrolysis was replaced by NY50C neutral protease and AX neutral protease, respectively.

[0058] In Comparative Example 4, replacing the 3.350 acidic enzyme used in the second hydrolysis with 3.4310 acidic enzyme did not result in any tyrosinase inhibition. In Comparative Example 5, replacing the 3.350 acidic enzyme with 537 acidic enzyme resulted in only a 7.63% inhibition of tyrosinase. Therefore, only by pretreating with aminopeptidase followed by sequential hydrolysis with PC10F neutral enzyme and 3.350 acidic enzyme can mulberry leaf polypeptides with high tyrosinase inhibitory activity be obtained.

[0059] Example 2

[0060] S220. Raw material pretreatment: Take 300g of crushed mulberry leaf powder (protein content: 17.64%), add 2700ml of purified water at 70℃, and soak for 40min.

[0061] S220, aminopeptidase pretreatment: Add 3g of aminopeptidase to the above soaking solution, adjust the pH value to 5.5 with hydrochloric acid, hydrolyze at 45℃ for 35min; then heat to 90℃ and keep warm for 1 hour, pour into a colloid mill while hot and homogenize for 1.5min to obtain a homogenized solution.

[0062] S230, First hydrolysis: Take the homogenate, add 211,680 U of PC10F neutral protease (Geobacillus stearothermophilus, Amano Amano Enzyme Preparation Trading Co., Ltd.) according to the protein content of the raw material, adjust the pH value to 7.5 with sodium hydroxide, and hydrolyze at a constant temperature of 55℃ for 60 min; after hydrolysis, heat to 90℃ for 9 minutes to inactivate the enzyme, and obtain the first hydrolysate.

[0063] S240, Second hydrolysis: After cooling the first hydrolysate obtained in step S230, add 158,760 U of 3,350 acidic protease (produced by Aspergillus niger strain) according to the protein content of the raw material. Adjust the pH to 3.0 with hydrochloric acid and keep it at 45°C for 50 min for hydrolysis. After the hydrolysis is completed, heat to 90°C for 4 min to inactivate the enzyme and obtain the second hydrolysate.

[0064] S250. Impurity Removal and Membrane Separation: The second hydrolysate obtained in step S240 is centrifuged at 5000 rpm for 8 min. The centrifuged liquid is then ultrafiltered using a 3000 Da ultrafiltration membrane to obtain polypeptide solutions with molecular weights greater than 3000 Da and less than 3000 Da. The polypeptide solutions with molecular weights less than 3000 Da are then ultrafiltered using a 1000 Da ultrafiltration membrane to obtain mulberry leaf polypeptides with molecular weights of 1000 Da to 3000 Da and less than 1000 Da.

[0065] The above peptide samples were adjusted to a peptide concentration of 15 mg / ml, and their tyrosinase inhibition rate was measured. The test results are shown in Table 2.

[0066] Table 2

[0067] polypeptide segments >3000Da 1000-3000Da <1000Da Tyrosinase activity inhibition rate % 46.23％ 48.06％ 71.2％

[0068] As can be seen from the test results in Table 2, the mulberry leaf peptides in this embodiment showed the highest inhibition rate against tyrosinase in peptides with a molecular weight below 1000 Da, with an inhibition rate of 71.2%. Peptides with a molecular weight greater than 3000 Da and between 1000 and 3000 Da also showed inhibition rates against tyrosinase, at 46.23% and 48.06%, respectively. This indicates that peptides with a molecular weight less than 1000 Da have the highest inhibitory effect on tyrosinase.

[0069] Example 3

[0070] S310. Raw material pretreatment: Take 600g of crushed mulberry leaf powder (protein content: 17.64%), add 5400ml of purified water at about 75℃, and soak for 70min.

[0071] S320, aminopeptidase pretreatment: Add 6g of aminopeptidase to the above soaking solution, adjust the pH value to 6.0 with hydrochloric acid, hydrolyze at 50℃ for 50min; then heat to 90℃ and keep warm for 2 hours, pour into a colloid mill while hot and homogenize for 2min to obtain a homogenized solution.

[0072] S330, First hydrolysis: Take the homogenate, add 635,040 U of PC10F neutral protease (Amano Amano Enzyme Preparation Trading Co., Ltd., Geobacillus stearothermophilus) according to the protein content of the raw material, adjust the pH value to 8.0 with sodium hydroxide, and hydrolyze at a constant temperature of 60℃ for 70 min; after hydrolysis, heat to 90℃ for 8 minutes to inactivate the enzyme, and obtain the first hydrolysate.

[0073] S340, Second hydrolysis: After cooling the first hydrolysate obtained in step S330, add 317,520 U of 3,350 acidic protease (produced by Aspergillus niger strain) according to the protein content of the raw material. Adjust the pH to 3.5 with hydrochloric acid and keep it at 50°C for 80 min for hydrolysis. After the hydrolysis is completed, heat to 90°C for 5 min to inactivate the enzyme and obtain the second hydrolysate.

[0074] S350. Impurity removal and membrane separation: Centrifuge the second hydrolysate obtained in step S340 at 6000 rpm for 10 min, and then ultrafilter the centrifuged liquid with a 1000 Da ultrafiltration membrane to obtain mulberry leaf polypeptide ultrafiltrate with a content of less than 1000 Da.

[0075] S360. In vitro simulated gastrointestinal digestion: Take 50 ml of the mulberry leaf polypeptide ultrafiltrate obtained in step S350 and 0.2 g NaCl. Adjust the pH to 2.0 with 1.0 mol / L HCl. Incubate in a water bath at 37°C. Add 0.05 g pepsin for 2 h. After cooling to room temperature, adjust the pH to 7.5 with 1.0 mol / L NaOH solution. Add 0.68 g KH2PO4. Incubate in a water bath at 37°C. Add 0.05 g trypsin for 4 h of constant temperature digestion. After digestion is complete, inactivate the enzymes in a boiling water bath at 100°C. After cooling to room temperature, add deionized water to make up to 100 mL.

[0076] It should be noted that the above-mentioned experimental method for simulating gastrointestinal digestion in vitro was based on the article "Antioxidant effect of pea oligopeptides in simulated gastrointestinal digestion system" published in the journal "Grains and Oils" Volume 3, Issue 4, 2021.

[0077] Comparative Example 6

[0078] The only difference from Example 3 is that pepsin and trypsin are not added in the in vitro simulated gastrointestinal digestion. All other operations and processes are the same as in Example 3.

[0079] The peptide concentration of the samples treated with gastrointestinal fluid was adjusted to 15 mg / ml, and the inhibition rate of tyrosinase was detected. The test results are shown in Table 3.

[0080] Table 3

[0081] Example 3 Comparative Example 6 Tyrosinase inhibition rate % 75.2％ 65.78％

[0082] As shown in Table 3, Comparative Example 6 differed from Example 3 only in that it did not include pepsin and trypsin in the in vitro simulated gastrointestinal digestion process; all other operations and processes were identical. Therefore, Comparative Example 6 can serve as a blank control for Example 3, eliminating the possibility that NaCl, KH₂PO₄, and constant-temperature water bath altered tyrosinase inhibitory activity. The experimental results revealed that after in vitro simulated gastrointestinal digestion, the mulberry leaf polypeptide not only exhibited anti-digestive properties but also enhanced tyrosinase inhibition. Compared to the untreated product, the inhibition of tyrosinase activity increased by 9.42%. This may be because the mulberry leaf polypeptide facilitates the release of peptides with higher tyrosinase inhibition rates during gastrointestinal digestion. Therefore, the mulberry leaf polypeptide prepared in this invention has the potential to be formulated into an oral product, allowing its whitening effect to be better realized.

[0083] In summary, the mulberry leaf polypeptides obtained by sequential enzymatic hydrolysis with PC10F neutral protease and 3.350 acidic protease in this invention exhibit significant inhibition of tyrosinase activity within a specific molecular weight range. This can achieve a cosmetic effect by inhibiting melanin production. Furthermore, it was unexpectedly discovered that the mulberry leaf polypeptides prepared in this invention, after in vitro digestion with simulated gastrointestinal fluid, not only showed resistance to digestion but also enhanced the inhibition of tyrosinase. This indicates that the whitening effect of these mulberry leaf polypeptides is likely to be better exerted after digestion in the human gastrointestinal tract.

[0084] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing mulberry leaf active polypeptides, characterized in that, The preparation steps include the following: First, mulberry leaves are crushed and soaked in water. Then, aminopeptidase is added for pretreatment and homogenization. Next, PC10F neutral protease and 3.350 acidic protease are added sequentially for two hydrolysis. Finally, the mulberry leaf hydrolysate obtained after two hydrolysis is centrifuged and passed through a 1000 Da ultrafiltration membrane to obtain mulberry leaf polypeptides with a molecular weight of less than 1000 Da. The amount of aminopeptidase added is 0.05% to 0.5% of the mass of the liquid in which it is added, and the treatment conditions of the aminopeptidase are pH value to 5.0 to 6.5 and hydrolysis at 40 to 55°C for 30 to 50 minutes. The pretreated solution obtained after aminopeptidase treatment needs to be heated to 70-90°C and kept at that temperature for 0.5-3 hours before homogenization for 1-2 minutes. The hydrolysis conditions for the PC10F neutral protease are constant temperature hydrolysis at pH 7.0–8.5 and 50–65°C for 50–100 min; The hydrolysis conditions for the 3.350 acidic protease are: constant temperature hydrolysis at pH 2.5–3.5 and 40–50°C for 40–80 min. Based on the protein content of the raw materials, the amount of PC10F neutral protease added is 3000-7000 U / g; Based on the protein content of the raw materials, the amount of the 3.350 acidic protease added is 3000-7000 U / g.

2. The method for preparing mulberry leaf active polypeptides according to claim 1, characterized in that: The mulberry leaves and water are mixed and soaked at a mass ratio of 1:6 to 9, and the soaking time is 30 to 90 minutes.

3. The method for preparing mulberry leaf active polypeptides according to claim 1, characterized in that: The hydrolysate obtained after hydrolysis of PC10F neutral protease needs to be heated to 90°C for 8-10 minutes to inactivate the enzyme. The hydrolysate obtained after hydrolysis of the 3.350 acidic protease needs to be heated to 90°C for 3-5 minutes to inactivate the enzyme.

4. The method for preparing mulberry leaf active polypeptide according to claim 1, characterized in that: The mulberry leaf hydrolysate, after two peptidase hydrolysis processes, was centrifuged at 4000–6000 rpm for 5–10 min and then ultrafiltered through a 1000 Da ultrafiltration membrane to obtain mulberry leaf peptides with a molecular weight of less than 1000 Da.

5. The method for preparing mulberry leaf active polypeptide according to claim 1, characterized in that: When mulberry leaf polypeptides are dried into powder and diluted to a concentration of 0.5%–2.0%, their tyrosinase inhibition rate reaches 20.6%–76.71%.

6. A mulberry leaf active polypeptide, characterized in that: It is prepared by the method of any one of claims 1 to 5 for the preparation of mulberry leaf active polypeptide.

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