A kind of guanshan cherry peptide with antioxidant and tyrosinase inhibitory activity and its preparation method

Abstract

This invention relates to the technical field of deep processing of Kanzan cherry blossoms, and particularly to a Kanzan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities and its preparation method. The preparation method includes the following steps: forming a first solution by mixing Kanzan cherry blossom powder with water; sequentially adding aminopeptidase, phytase, and pectinase to the first solution for hydrolysis pretreatment, followed by homogenization; sequentially adding AX neutral protease and 3.4310 acidic protease to the homogenized solution for two hydrolysis processes to obtain a hydrolysate; separating the solid and liquid components of the hydrolysate, and filtering the supernatant to retain peptides with a molecular weight cutoff of less than 1000 Da. Using this invention, Kanzan cherry blossom peptides and their products possessing tyrosinase inhibitory activity, antioxidant activity, and probiotic effects can be obtained.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of Kanzan cherry blossoms, and in particular to a Kanzan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities and its preparation method. Background Technology

[0002] Cherry blossoms are widely planted in Kanzan. Because they are rich in nutrients such as polysaccharides and proteins, they are mainly used in the following ways: eating them fresh directly, adding them to baked goods after drying, grinding the petals into nectar to make jams and condiments, and using them as ornamental plants. However, there are not many deep-processed products made from them.

[0003] There are few research reports and patents on Kanzan cherry blossoms; research mainly focuses on volatile oils, polysaccharides and their extracts, and flavonoids in cherry blossoms. For example:

[0004] In their article "Antioxidant and Anti-inflammatory Effects of Cherry Blossom", Wang Ruirui et al. mentioned that they obtained an antioxidant extract of Cherry Blossom using supercritical carbon dioxide extraction. However, this method has high requirements for raw materials and equipment, and it is costly and difficult to achieve large-scale production.

[0005] In Chinese invention patent application document with application number CN202111022447.5 and publication date of December 21, 2021, a cherry blossom enzymatic hydrolysis fermentation product and its preparation method and application are disclosed. The method uses enzyme solution to treat cherry blossoms and then ferments them with lactic acid bacteria and yeast to obtain an antioxidant fermentation liquid with high flavonoid content, but does not involve the activity detection of cherry blossom peptides.

[0006] Therefore, existing technologies mainly focus on the study of volatile oils, polysaccharides and their extracts, and flavonoids in cherry blossoms, while the field of peptide extraction and activity research of Kanzan cherry blossoms remains largely unexplored. Existing literature and patents have not reported on the preparation of bioactive peptides with tyrosinase inhibitory activity, antioxidant properties, and probiotic effects using Kanzan cherry blossoms. Summary of the Invention

[0007] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities, the technical solution of which is as follows:

[0008] The first liquid is made by mixing Kanzan cherry blossom powder with water;

[0009] After hydrolysis pretreatment by sequentially adding aminopeptidase, phytase and pectinase to the first material solution, homogenization is performed to obtain a homogenized solution.

[0010] The homogenate was hydrolyzed twice by adding AX neutral protease and 3.4310 acidic protease in sequence to obtain the hydrolysate;

[0011] The hydrolysate is separated into solid and liquid components, and the supernatant is filtered to retain the polypeptide, thus obtaining the Guanshan cherry blossom peptide.

[0012] In one embodiment, the hydrolysate is separated into solid and liquid components, and the supernatant is filtered to retain polypeptides with a molecular weight of less than 1000 Da, thus obtaining the final product.

[0013] In one embodiment, the hydrolysis pretreatment includes the following steps:

[0014] Based on the mass of the first liquid, add 0.1% to 0.3% of the aminopeptidase to the first liquid, adjust the pH to 4.0 to 6.0, and hydrolyze at 30 to 60°C for 50 to 90 minutes to obtain the first treatment solution;

[0015] Based on the mass of the first treatment solution, add 0.1% to 0.3% phytase to the first treatment solution, adjust the pH value to 3.5 to 5.0, and hydrolyze at 40 to 60°C for 50 to 90 minutes to obtain the second treatment solution;

[0016] Based on the mass of the Guanshan cherry blossom powder raw material, 0.5% to 2.0% pectinase was added to the second treatment solution, the pH value was adjusted to 3.5 to 5.0, and hydrolyzed at 40 to 60°C for 30 to 80 minutes to obtain the third treatment solution.

[0017] In one embodiment, the homogenized liquid undergoes two hydrolysis processes, including the following steps:

[0018] The homogenate was added with neutral protease AX, and after adjusting the pH, a first hydrolysis was performed; then acidic protease 3.4310 was added, and after adjusting the pH, a second hydrolysis was performed.

[0019] The conditions for the first hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 6.0-7.5 and a temperature of 40-60℃; the conditions for the second hydrolysis are: constant temperature hydrolysis for 50-90 min at a system pH of 2.0-3.5 and a temperature of 40-60℃.

[0020] In one embodiment, the amount of neutral protease AX added is 4000-9000 U / g, calculated based on the protein content of the raw material of Kanzan cherry blossom powder; and the amount of acidic protease 3.4310 added is 3000-8000 U / g, calculated based on the protein content of the raw material of Kanzan cherry blossom powder.

[0021] In one embodiment, the hydrolysate obtained after the first hydrolysis is subjected to a first enzyme inactivation treatment, and then subjected to a second hydrolysis; the hydrolysate obtained after the second hydrolysis is subjected to a second enzyme inactivation treatment to obtain a hydrolysate.

[0022] In one embodiment, the hydrolysate obtained after the first hydrolysis is heated to 80°C to 90°C for 10 to 20 minutes to inactivate the enzyme, and then subjected to a second hydrolysis; the hydrolysate obtained after the second hydrolysis is heated to 80°C to 90°C for 5 to 15 minutes to inactivate the enzyme, thus obtaining the hydrolysate.

[0023] In one embodiment, after centrifuging the hydrolysate, the supernatant is subjected to ultrafiltration with a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da.

[0024] In one embodiment, pulverized Kanzan cherry blossoms are added to water and soaked at 50–80°C for 1–2 hours to form a first liquid; wherein the mass ratio of the Kanzan cherry blossom powder to the water is 1:(16–20).

[0025] In one embodiment, the pre-treated hydrolysis solution is homogenized in a colloid mill for 2-3 minutes to obtain a homogenized solution;

[0026] In one embodiment, the hydrolysate is centrifuged at 3000-8000 rpm for 5-15 min to perform solid-liquid separation.

[0027] The present invention also provides a Kanzan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities, which is prepared by the Kanzan cherry blossom peptide preparation method described above.

[0028] The present invention also provides a food product comprising Kanzan cherry blossom peptide; the Kanzan cherry blossom peptide is prepared by the preparation method of Kanzan cherry blossom peptide as described above.

[0029] Based on the above, compared with the prior art, the preparation method of Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities of the present invention has the following beneficial effects:

[0030] This invention can be used to prepare polypeptides that possess tyrosinase inhibitory activity, antioxidant activity, and probiotic effects.

[0031] (1) In this invention, after sequentially treating the cherry blossoms with aminopeptidase, phytase and pectinase, the cherry blossoms are sequentially hydrolyzed by AX neutral protease and 3.4310 acidic protease to obtain the cherry blossom peptide. The peptide has the properties of inhibiting tyrosinase activity and antioxidant activity.

[0032] (2) The polypeptide obtained in this invention has a promoting effect on the proliferation of Lactobacillus paracasei YYS-69 (CGMCC NO.25837), Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and Lactobacillus fermentum YYS-K2 (CGMCC NO.27129);

[0033] (3) The Guanshan cherry blossom peptide prepared by the present invention has high temperature resistance. After high temperature treatment, it has no bitter taste and good flavor. When it is applied to food, it has a good taste and flavor, which is conducive to improving the user experience.

[0034] (4) Simple process: The method of the present invention can obtain the required peptides by using a simple combination of operations such as enzymatic hydrolysis, homogenization and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0035] Other features and advantages 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. Detailed Implementation

[0036] 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 some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. 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.

[0037] 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.

[0038] This invention provides an operational example of a method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities, the specific steps of which are as follows:

[0039] S100. Raw material pretreatment: After crushing the cherry blossoms of Guanshan, add them to pure water at 50-80℃ at a mass ratio of 1:(16-20) and soak for 1-2 hours.

[0040] S200, aminopeptidase treatment: Based on the mass of the soaking solution obtained in step S100, add 0.1% to 0.3% aminopeptidase, adjust the pH value to 4.0 to 6.0 with hydrochloric acid, and hydrolyze at 30 to 60°C for 50 to 90 minutes.

[0041] S300, Phytase Treatment: Obtain the hydrolysate according to step S200, add 0.1% to 0.3% phytase by weight of the liquid, adjust the pH to 3.5 to 5.0 with hydrochloric acid, and hydrolyze at 40 to 60°C for 50 to 90 minutes.

[0042] S400, pectinase treatment: Based on the weight of Guanshan cherry blossom powder raw material, add 0.5% to 2.0% pectinase, and the pectinase treatment conditions are pH value to 3.5 to 5.0, hydrolysis at 40 to 60℃ for 30 to 80 minutes.

[0043] S500. The treatment solution obtained after treatment with aminopeptidase, phytase and pectinase is homogenized in a colloid mill for 2-3 minutes.

[0044] S600, First hydrolysis: Calculate based on the protein content of the raw material, add 4000-9000 U / g of neutral proteinase AX to the homogenized solution, adjust the pH value to 6.0-7.5 with sodium hydroxide solution, and hydrolyze at a constant temperature of 40-60℃ for 60-90 min. After hydrolysis, heat to 80-90℃ for 10-20 min to inactivate the enzyme, and obtain the first hydrolysate.

[0045] S700, Second hydrolysis: Based on the protein content of the raw material, after cooling the first hydrolysate obtained in step S400, add 3000-8000 U / g of 3.4310 acidic protease, adjust the pH to 2.0-3.5 with hydrochloric acid, and keep it at 40-60℃ for 50-90 min for hydrolysis. After hydrolysis, heat to 80-90℃ for 5-15 min to inactivate the enzyme and obtain the second hydrolysate.

[0046] S800, Impurity Removal and Membrane Separation: The second hydrolysate obtained in step S500 is centrifuged at 3000-8000 rpm for 5-15 min, and then the supernatant is passed through a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da, which is then spray-dried into powder for storage.

[0047] The present invention provides the following embodiments.

[0048] Example 1

[0049] S110. Raw material pretreatment: Take 300g of crushed Guanshan cherry blossom powder (protein content: 23.4%), add 4800ml of 50℃ pure water, and soak for 60min.

[0050] S120, aminopeptidase treatment: Add 5.1g of aminopeptidase to the above soaking solution, adjust the pH value to 4.5 with hydrochloric acid, and hydrolyze at 35℃ for 50min.

[0051] S130, Phytase Treatment: Obtain the hydrolysate according to step S120, add 10.2g of phytase, adjust the pH to 3.5 with hydrochloric acid, and hydrolyze at 40℃ for 50min.

[0052] S140, Pectinase Treatment: Add 1.5g of pectinase, and the pectinase treatment conditions are pH value to 4.0 and hydrolysis at 40℃ for 30min.

[0053] S150. The treatment solution obtained after treatment with aminopeptidase, phytase and pectinase is homogenized in a colloid mill for 2 min.

[0054] S160, First hydrolysis: Take the pretreated liquid, add 280,800 U of neutral protein (AX) according to the protein content of the raw material, adjust the pH value to 6.0 with sodium hydroxide, and hydrolyze at a constant temperature of 50℃ for 60 min; after hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme, and obtain the first hydrolysate.

[0055] S170, Second hydrolysis: After cooling the first hydrolysate obtained in step S170, add 210,600 U of 3,4310 acidic protease according to the protein content of the raw material, adjust the pH to 2.5 with hydrochloric acid, keep at 40℃ for 50 min for hydrolysis, and heat to 90℃ for 5 min to inactivate the enzyme to obtain the second hydrolysate.

[0056] S180. Impurity removal and membrane separation: The second hydrolysate obtained in step S180 is centrifuged at 4000 rpm for 5 min, and then ultrafiltered through a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da, and then spray-dried into powder for storage.

[0057] The results showed that when the polypeptide powder prepared in Example 1 was diluted with pure water to a polypeptide content of 10 mg / ml, the inhibition rate of tyrosinase was 41.3%; when diluted to a polypeptide content of 15 mg / ml, the inhibition rate of tyrosinase was 62.5%; and when diluted to a polypeptide content of 20 mg / ml, the inhibition rate of tyrosinase was 76.2%.

[0058] It should be noted that the method for testing the inhibition rate of tyrosinase in this invention adopts T / SHRH 015~2018 Cosmetics~Tyrosinase Activity Inhibition Test.

[0059] To better illustrate that the preparation method provided by the present invention can obtain Guanshan cherry blossom peptide with high tyrosinase inhibitory activity, the present invention also provides the following comparative examples 1 to 10:

[0060] Comparative Example 1

[0061] The only difference from Example 1 is that the aminopeptidase, phytase, pectinase, AX neutral protease, and 3.4310 acidic protease are not added. All other operations and process conditions are the same as in Example 1.

[0062] Comparative Example 2

[0063] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with aminopeptidase; all other operations and process conditions are the same as in Example 1.

[0064] Comparative Example 3

[0065] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with phytase; all other operations and process conditions are the same as in Example 1.

[0066] Comparative Example 4

[0067] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with pectinase; all other operations and process conditions are the same as in Example 1.

[0068] Comparative Example 5

[0069] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with aminopeptidase and phytase. All other operations and process conditions are the same as in Example 1.

[0070] Comparative Example 6

[0071] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with aminopeptidase and pectinase. All other operations and process conditions are the same as in Example 1.

[0072] Comparative Example 7

[0073] The only difference from Example 1 is that the soaking solution obtained in step S100 was not treated with phytase and pectinase. All other operations and process conditions are the same as in Example 1.

[0074] Comparative Example 8

[0075] The only difference from Example 1 is that papain and other enzymes are used to replace the neutral AX protease, and the papain hydrolysis conditions are 60°C and pH 6.5. All other operations and process conditions are the same as in Example 1.

[0076] Comparative Example 9

[0077] The only difference from Example 1 is that 2.4L of alkaline protease is used instead of neutral protease AX, and the hydrolysis conditions of NY50C protease are 55°C and pH 8.5. All other operations and process conditions are the same as in Example 1.

[0078] Comparative Example 10

[0079] The only difference from Example 1 is that 3.4310 acidic protease is replaced with enzymes such as 537 acidic protease, 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.

[0080] It should be noted that, since the enzymes used in the above (e.g., comparative examples 8-10) have different applicable pH levels, the pH of the solutions in the comparative examples was also adjusted accordingly to suit the use of different enzymes. The limiting role of the environmental pH value is to adapt to the enzyme's usage conditions.

[0081] The peptide samples obtained above were prepared into solutions with a peptide concentration of 15 mg / ml using pure water, and their tyrosinase inhibition rate was detected. The test results are shown in Table 1.

[0082] Table 1

[0083]

[0084] Note: - Not detected

[0085] The test results in Table 1 show that:

[0086] (1) In Example 1, after pretreatment with aminopeptidase, phytase, and pectinase, followed by hydrolysis inhibition by AX neutral protease and 3.4310 acidic protease, the inhibition rate reached as high as 62.5%;

[0087] (2) In Comparative Example 1, no tyrosinase inhibition rate was detected in the hydrolysate without enzyme treatment;

[0088] (3) Comparative Examples 2, 3, 4, 5, 6, and 7 were treated with one or two of aminopeptidase, phytase, and pectinase, and then hydrolyzed with AX neutral protease and 3.4310 acidic protease. Compared with Example 1, the inhibition rate of the prepared peptides against tyrosinase decreased by 47.5%, 52.3%, 46.2%, 53.8%, 56.1%, and 49.6%, respectively. The reason for this may be that aminopeptidase opens the binding between protein and cellulose, phytase opens the binding between protein and minerals, and the pectinase treatment reduces the viscosity of the hydrolysate of the cherry blossoms, increasing the hydrolysis yield and making the hydrolysis more thorough, thereby releasing more active peptides.

[0089] (4) Comparative Example 8 showed no inhibitory effect on tyrosinase when papain was used to replace the neutral AX protease required for the first hydrolysis.

[0090] (5) Comparative Example 9 replaced the AX neutral protease required for the first hydrolysis with 2.4L alkaline protease and found that it had a small inhibitory effect on tyrosinase, which was 3.2%.

[0091] (6) In Comparative Example 10, the 3.4310 acidic protease used in the second hydrolysis was replaced with 537 acidic enzyme, and no inhibitory effect of tyrosinase was detected.

[0092] In summary, only by first pretreating with aminopeptidase, phytase, and pectinase, and then sequentially hydrolyzing with AX neutral enzyme and 3.4310 acidic protease, can Guanshan cherry blossom peptide with high tyrosinase inhibitory activity be obtained.

[0093] Example 2

[0094] S220. Raw material pretreatment: Take 200g of crushed Guanshan cherry blossom powder (protein content: 23.4%), add 3400ml of 60℃ pure water, and soak for 90min.

[0095] S220, aminopeptidase treatment: Add 3.6g of aminopeptidase to the above soaking solution, adjust the pH value to 5.0 with hydrochloric acid, and hydrolyze at 50℃ for 60min.

[0096] S230, Phytase Treatment: Obtain the hydrolysate according to step S220, add 7.2g of phytase, adjust the pH to 4.0 with hydrochloric acid, and hydrolyze at 45℃ for 60min.

[0097] S240, Pectinase Treatment: Add 2g of pectinase, and the pectinase treatment conditions are pH value to 4.5 and hydrolysis at 50℃ for 40min.

[0098] S250. The treatment solution obtained after treatment with aminopeptidase, phytase and pectinase is homogenized in a colloid mill for 1.5 min.

[0099] S260, First hydrolysis: Take the pretreated liquid, add 234,000 U of AX neutral protease according to the protein content of the raw material, adjust the pH value to 6.5 with sodium hydroxide, and hydrolyze at a constant temperature of 55℃ for 70 min; after hydrolysis, heat to 80℃ for 20 min to inactivate the enzyme, and obtain the first hydrolysate.

[0100] S270, Second hydrolysis: After cooling the first hydrolysate obtained in step S260, add 187,200 U of 3.4310 acidic protease according to the protein content of the raw material, adjust the pH to 2.0 with hydrochloric acid, and hydrolyze at a constant temperature of 50℃ for 60 min. After the hydrolysis is completed, heat to 85℃ to inactivate the enzyme for 10 min to obtain the second hydrolysate.

[0101] S280. Impurity removal and membrane separation: Centrifuge the second hydrolysate obtained in step S270 at 6000 rpm for 10 min.

[0102] The centrifuged liquid was ultrafiltered using a 5000 Da ultrafiltration membrane to obtain peptide solutions with molecular weights greater than 5000 Da and less than 5000 Da. The peptide solutions with molecular weights less than 5000 Da were then ultrafiltered using a 3000 Da ultrafiltration membrane to obtain peptides with molecular weights of 3000 Da to 5000 Da and less than 3000 Da. Finally, the peptide solutions with molecular weights less than 3000 Da were ultrafiltered using a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptides with molecular weights of 1000 Da to 3000 Da and less than 1000 Da.

[0103] The above peptide samples were prepared into solutions with a peptide concentration of 15 mg / ml using pure water, and their tyrosinase inhibition rate was measured. The test results are shown in Table 2.

[0104] Table 2

[0105]

[0106] The test results in Table 2 show that:

[0107] (1) The Guanshan cherry blossom peptide in Example 2 showed the highest inhibition rate of tyrosinase in the polypeptide segment below 1000 Da, with an inhibition rate of 65.4%;

[0108] (2) Polypeptide solutions with molecular weights greater than 5000 Da, 3000–5000 Da, and 1000–3000 Da also showed inhibition rates of tyrosinase, at 15.7%, 23.6%, and 34.8%, respectively.

[0109] This shows that peptides with a molecular weight of less than 1000 Da have the highest inhibitory effect on tyrosinase.

[0110] Example 3

[0111] S310. Raw material pretreatment: Take 500g of crushed Guanshan cherry blossom powder (protein content: 23.4%), add 10000ml of 80℃ pure water, and soak for 100min.

[0112] S320, aminopeptidase treatment: Add 10.5g of aminopeptidase to the above soaking solution, adjust the pH value to 6.0 with hydrochloric acid, and hydrolyze at 55℃ for 90min.

[0113] S330, Phytase Treatment: Obtain the hydrolysate according to step S320, add 10.5g of phytase, adjust the pH to 4.7 with hydrochloric acid, and hydrolyze at 50℃ for 80min.

[0114] S340, Pectinase treatment: The hydrolysate obtained in step S330 is added with 2.5g of pectinase, and the pectinase treatment conditions are pH value to 4.7 and hydrolysis at 55℃ for 70min.

[0115] S350. The treatment solution obtained after treatment with aminopeptidase, phytase and pectinase is homogenized in a colloid mill for 2 min.

[0116] S360, First hydrolysis: Take the pretreated liquid, add 819,000 U of AX neutral protease according to the protein content of the raw material, adjust the pH value to 7.0 with sodium hydroxide, and hydrolyze at a constant temperature of 60℃ for 80 min; after hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme, and obtain the first hydrolysate.

[0117] S370, Second hydrolysis: After cooling the first hydrolysate obtained in step S360, add 819,000 U of 3,4310 acidic protease according to the protein content of the raw material, adjust the pH to 3.0 with hydrochloric acid, hydrolyze at a constant temperature of 60℃ for 80 min, and heat to 85℃ to inactivate the enzyme for 10 min to obtain the second hydrolysate.

[0118] S380. Impurity removal and membrane separation: The second hydrolysate obtained in step S370 is centrifuged at 7000 rpm for 15 min, and then ultrafiltered through a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da, and then spray-dried into powder for storage.

[0119] Upon testing, when the polypeptide powder obtained in Example 3 was prepared with pure water to a polypeptide content of 15 mg / ml, the DPPH scavenging rate, ABTS scavenging rate, and hydroxyl radical scavenging rate were simultaneously tested. The DPPH scavenging rate reached 93.3%, the ABTS scavenging rate reached 98.7%, and the hydroxyl radical scavenging rate reached 96.3%. It should be noted that the test methods for DPPH scavenging rate and ABTS scavenging rate in this invention are GB / T39100-2020, and the test method for hydroxyl radical scavenging rate is the salicylic acid method.

[0120] In addition, the peptide concentration of the samples in Example 3 and Comparative Examples 1-10 was adjusted to 15 mg / ml, and the antioxidant index was detected. The test results are shown in Table 3.

[0121] Table 3

[0122]

[0123] Note: - Not detected

[0124] The results in Table 3 show that:

[0125] (1) In Example 3, the Guanshan cherry blossom peptide, which was pretreated with aminopeptidase, phytase, pectinase, and AX neutral protease and treated with 3.4310 acidic protease, showed an antioxidant index of 93.3% DPPH scavenging rate, 98.7% ABTS scavenging rate, and 96.3% hydroxyl radical scavenging rate.

[0126] (2) The water extract obtained in Comparative Example 1 without enzyme treatment has certain antioxidant properties, but its antioxidant properties are lower than those of the Guanshan cherry blossom peptide obtained by pretreatment with aminopeptidase, phytase, pectinase and hydrolysis with AX neutral protease and 3.4310 acidic protease. The DPPH, ABTS and hydroxyl radical scavenging rates are 40.9%, 45.2% and 31.5% lower, respectively.

[0127] The reason for this phenomenon may be that the effective antioxidant components are better hydrolyzed after the addition of protease. At the same time, the experimental group with added Kanzan cherry blossom peptides immediately decolorized the reaction substrate, which also better illustrates the strong antioxidant properties of Kanzan cherry blossoms.

[0128] (3) Comparative Examples 2-7 were treated with one or two of aminopeptidase, phytase, and pectinase, and then hydrolyzed by AX neutral protease and 3.4310 acidic protease. Their antioxidant capacity was significantly lower than that of the Examples.

[0129] (4) Comparative Examples 8-10 used other enzymes to replace AX neutral protease or 3.4310 acidic protease for enzymatic hydrolysis, and their antioxidant properties were significantly lower than those of the Examples.

[0130] Example 4

[0131] S410. Raw material pretreatment: Take 600g of crushed Guanshan cherry blossom powder (protein content: 23.4%), add 11400ml of 80℃ pure water, and soak for 120min.

[0132] S420, aminopeptidase treatment: Add 12g of aminopeptidase to the above soaking solution, adjust the pH value to 4.0 with hydrochloric acid, and hydrolyze at 40℃ for 90min.

[0133] S430, Phytase Treatment: Obtain the hydrolysate according to step S420, add 22.8g of phytase, adjust the pH to 5.0 with hydrochloric acid, and hydrolyze at 55℃ for 90min.

[0134] S440, Pectinase Treatment: The hydrolysate obtained in step S430 is added to 3.0g of pectinase, and the pectinase treatment conditions are pH value to 5.0 and hydrolysis at 60℃ for 80min.

[0135] S450. The treatment solution obtained after treatment with aminopeptidase, phytase and pectinase is homogenized in a colloid mill for 1 min.

[0136] S460, First hydrolysis: Take the pretreated liquid, add 1,123,200 U of neutral proteinase AX 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 60℃ for 90 min; after hydrolysis, heat to 90℃ for 16 min to inactivate the enzyme, and obtain the first hydrolysate.

[0137] S470, Second hydrolysis: After cooling the first hydrolysate obtained in step S460, add 982,800 U of 3.4310 acidic protease according to the protein content of the raw material, adjust the pH to 3.5 with hydrochloric acid, hydrolyze at a constant temperature of 60℃ for 90 min, and heat to 85℃ to inactivate the enzyme for 15 min to obtain the second hydrolysate.

[0138] S480. Impurity removal and membrane separation: The second hydrolysate obtained in step S470 is centrifuged at 8000 rpm for 15 min, and then ultrafiltered through a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da, and then spray-dried into powder for storage.

[0139] S490. Sterilization: Prepare a 15 mg / ml polypeptide solution from the Guanshan cherry blossom peptide obtained in step S480, and inactivate the enzyme at 121℃ for 25 min.

[0140] S491. Add 10 ml of the peptide solution containing 15 mg / ml of Guanshan cherry blossom peptide obtained in step S490 to 50 ml of MRS medium. The peptide solution was tested and found to have a 13% proliferation rate for Lactobacillus paracasei YYS-69 (CGMCC NO.25837), a 21% proliferation rate for Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and a 16% proliferation rate for Lactobacillus fermentum YYS-K2 (CGMCC NO.27129).

[0141] The peptide concentration of the samples from Example 4 and Comparative Examples 1-10 was adjusted to 15 mg / ml, and the probiotic proliferation rate was detected. The test results are shown in Table 4.

[0142] Table 4

[0143]

[0144] Note: - Not detected

[0145] The proliferation rate in Table 4 is calculated as follows: (number of viable bacteria in the experimental group - number of viable bacteria in the blank group) / number of viable bacteria in the blank group × 100%. For the calculation of the number of viable bacteria, please refer to Zhang Xiaofan, Environmental Microbiology, Shanghai: Shanghai Jiaotong University Press, April 2013.

[0146] Sensory evaluation was performed on the sterilized samples of Example 4 and Comparative Examples 1-10 with a peptide concentration of 15 mg / ml. The test results are shown in Table 5.

[0147] Table 5

[0148]

[0149] Five professional sensory evaluators were selected to taste the product and evaluate its color, flavor, and texture.

[0150] 1. The results in Table 4 show that:

[0151] (1) In the example, the Guanshan cherry blossom peptide obtained by pretreatment with aminopeptidase, phytase and pectinase in sequence, followed by hydrolysis with AX neutral enzyme and 3.4310 acidic protease combination, showed the best proliferation effect on Lactobacillus paracasei YYS-69 (CGMCC NO.25837), Lactobacillus plantarum YYS-99 (CGMCC NO.25838) and Lactobacillus fermentum YYS-K2 (CGMCC NO.27129), with 13%, 21% and 16% respectively. Therefore, Guanshan cherry blossom peptide also has the effect of promoting intestinal digestion.

[0152] (2) The proliferation effects of Comparative Examples 1-10 (different enzymatic hydrolysis combinations) on Lactobacillus paracasei YYS-69 (CGMCC NO.25837), Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and Lactobacillus fermentum YYS-K2 (CGMCC NO.27129) were significantly less than those of the Examples.

[0153] 2. The results in Table 5 show that:

[0154] After being pretreated with aminopeptidase, phytase and pectinase in sequence, the Guanshan cherry blossom peptide was hydrolyzed by a combination of AX neutral enzyme and 3.4310 acidic protease. After high-temperature sterilization, the color, flavor and taste remained unchanged. Guanshan cherry blossom peptide has the advantages of high temperature resistance, no bitterness and good flavor. Guanshan cherry blossom peptide has a competitive advantage in terms of sensory perception and can be used in food (such as beverages).

[0155] In summary, the method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities provided by the present invention has at least the following mechanisms of action and technical effects:

[0156] (1) In this invention, after sequentially treating Guanshan cherry blossoms with aminopeptidase, phytase and pectinase, Guanshan cherry blossom peptides are obtained by sequentially enzymatically hydrolyzing them with AX neutral protease and 3.4310 acidic protease. These peptides have the ability to inhibit tyrosinase activity, especially in a specific molecular weight range (peptide segments below 1000 Da), which significantly inhibits tyrosinase activity and thus has the function of inhibiting melanin production, and can play a good whitening role; at the same time, it has good antioxidant properties.

[0157] As can be seen from the specific examples, when the peptide content is 10 mg / ml to 25 mg / ml, its tyrosinase inhibition rate can reach 40% to 80%. At the same time, when the peptide content is 15 mg / ml, the DPPH scavenging rate reaches 93.3%, the ABTS scavenging rate reaches 98.7%, and the hydroxyl radical scavenging rate reaches 96.3%.

[0158] (2) The polypeptide obtained in this invention promotes the proliferation of Lactobacillus paracasei YYS-69 (CGMCC NO.25837), Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and Lactobacillus fermentum YYS-K2 (CGMCC NO.27129), thereby also promoting intestinal absorption.

[0159] In the specific examples, it can be seen that adding 15 mg / ml of polypeptide solution to MRS medium promotes the proliferation of probiotics, resulting in a 13% proliferation rate for Lactobacillus paracasei YYS-69 (CGMCC NO.25837), a 21% proliferation rate for Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and a 16% proliferation rate for Lactobacillus fermentum YYS-K2 (CGMCC NO.27129).

[0160] (3) The Guanshan cherry blossom peptide prepared by the present invention has high temperature resistance. After high temperature treatment, it has no bitter taste and good flavor. When it is applied to food, it has a good taste and flavor, which is conducive to improving the user experience.

[0161] (4) Simple process: The method of the present invention can obtain the required peptides by using a simple combination of operations such as enzymatic hydrolysis, homogenization and filtration. It has low equipment requirements and the process is simple and easy to operate.

[0162] It should be noted that:

[0163] (1) Definition:

[0164] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0165] The term "food" as used herein is used in a broad sense, encompassing both human food and drink. In some embodiments, the food product is suitable for and designed for human consumption.

[0166] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0167] In the text, "ABTS" refers to 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0168] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0169] The term "U / g" used in this article refers to the International Unit of Enzyme Activity, which is the amount of enzyme required to convert 1 micromolar of substrate or 1 micromolar of related groups in a substrate within 1 minute under specific conditions. It is called one International Unit (IU, also known as U). Enzyme activity, also known as enzyme activity, refers to the ability of an enzyme to catalyze a certain chemical reaction.

[0170] The term "ultrafiltration" as used in this article is a commonly used name for a processing step in the field, and its name accurately describes the process, so it will not be repeated here.

[0171] (2) Raw materials used in implementation:

[0172] The aminopeptidase, phytase, pectinase, AX neutral protease, and 3.4310 acidic protease used are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0173] The Lactobacillus paracasei YYS-69 used Lactobacillus paracasei The accession was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25837.

[0174] Lactobacillus plantarum YYS-99 ( Lactiplantibacillus plantarum The accession was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25838.

[0175] Fermented Lactobacillus mucinus YYS-K2 ( Limosilactobacillus fermentum The strain was deposited on April 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.27129. Technicians can obtain the strain from the collection center based on the accession number.

[0176] (3) Application of Guanshan cherry blossom peptide:

[0177] Based on the property that Guanshan cherry blossom peptide can inhibit tyrosinase activity, and tyrosinase is a key enzyme in melanin synthesis, it has the function of inhibiting melanin production and can play a good whitening role. Based on this, Guanshan cherry blossom peptide can also be applied to products (such as food, health products, etc.) with other specific effects besides inhibiting melanin formation, based on its properties of inhibiting tyrosinase activity or antioxidant properties.

[0178] Based on the fact that Guanshan cherry blossom peptide can promote the proliferation of Lactobacillus paracasei YYS-69 (CGMCC NO.25837), Lactobacillus plantarum YYS-99 (CGMCC NO.25838), and Lactobacillus fermentum YYS-K2 (CGMCC NO.27129), it also has the function of promoting intestinal absorption;

[0179] Based on this, Guanshan cherry blossom peptide can also be applied to products (such as food, health products, etc.) with other significant effects besides promoting intestinal digestion and absorption, based on its characteristic of promoting the proliferation of probiotics.

[0180] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

[0181] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities, characterized in that... This includes the following steps: The cherry blossom powder from Kanzan is crushed and added to water, then soaked at 50-80℃ for 1-2 hours to form the first liquid; wherein the mass ratio of the Kanzan cherry blossom powder to the water is 1:(16-20); Aminopeptidase, phytase, and pectinase are added sequentially to the first feed solution for hydrolysis pretreatment. The pretreated solution is then homogenized in a colloid mill for 2–3 min to obtain a homogenized solution. The homogenate was hydrolyzed twice by adding AX neutral protease and 3.4310 acidic protease in sequence to obtain the hydrolysate; The hydrolysate was centrifuged at 3000-8000 rpm for 5-15 min to separate solids and liquids. The supernatant was filtered to retain polypeptides with a molecular weight of less than 1000 Da, which was then obtained. The hydrolysis pretreatment includes the following steps: Based on the mass of the first liquid, add 0.1% to 0.3% of the aminopeptidase to the first liquid, adjust the pH to 4.0 to 6.0, and hydrolyze at 30 to 60°C for 50 to 90 minutes to obtain the first treatment solution; Based on the mass of the first treatment solution, add 0.1% to 0.3% phytase to the first treatment solution, adjust the pH value to 3.5 to 5.0, and hydrolyze at 40 to 60°C for 50 to 90 minutes to obtain the second treatment solution; Based on the mass of the Guanshan cherry blossom powder raw material, 0.5% to 2.0% pectinase was added to the second treatment solution, the pH value was adjusted to 3.5 to 5.0, and hydrolyzed at 40 to 60°C for 30 to 80 minutes to obtain the third treatment solution; The homogenized solution undergoes two hydrolysis processes, including the following steps: The homogenized solution was treated with AX neutral protease to adjust the pH, followed by a first hydrolysis. Then, 3.4310 acidic protease was added, and after adjusting the pH, a second hydrolysis was performed. The first hydrolysis was conducted at a system pH of 6.0–7.5 and a temperature of 40–60°C for 60–90 minutes under constant temperature conditions. The second hydrolysis was conducted at a system pH of 2.0–3.5 and a temperature of 40–60°C for 50–90 minutes under constant temperature conditions. Based on the protein content of the Guanshan cherry blossom powder raw material, the amount of AX neutral protease added was 4000–9000 U / g; based on the protein content of the Guanshan cherry blossom powder raw material, the amount of 3.4310 acidic protease added was 3000–8000 U / g.

2. The method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities according to claim 1, characterized in that: The hydrolysate obtained after the first hydrolysis is heated to 80℃~90℃ for 10~20 minutes to inactivate the enzyme, and then subjected to a second hydrolysis. The hydrolysate obtained after the second hydrolysis is heated to 80℃~90℃ for 5~15 minutes to inactivate the enzyme, thus obtaining the hydrolysate.

3. The method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities according to claim 1, characterized in that: After centrifugation, the supernatant was subjected to ultrafiltration with a 1000 Da ultrafiltration membrane to obtain Guanshan cherry blossom peptide with a molecular weight of less than 1000 Da.

4. The method for preparing Guanshan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities according to claim 3, characterized in that: The cherry blossom powder from Kanzan is crushed and added to water, then soaked at 50-80℃ for 1-2 hours to form the first liquid; wherein the mass ratio of the Kanzan cherry blossom powder to the water is 1:(16-20); And / or, the pretreated hydrolysis solution is homogenized in a colloid mill for 2-3 minutes to obtain a homogenized solution; And / or, the hydrolysate is centrifuged at 3000-8000 rpm for 5-15 min to perform solid-liquid separation.

5. A Kanzan cherry blossom peptide with antioxidant and tyrosinase inhibitory activities, characterized in that: It was prepared by the method described in any one of claims 1-4 for the preparation of Guanshan cherry blossom peptide.

6. A food product, characterized in that: Its components include Guanshan cherry blossom peptide; The Guanshan cherry blossom peptide was prepared using the preparation method of Guanshan cherry blossom peptide as described in any one of claims 1-4.

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